Mitochondrial Permeability Transition Pore Inhibitors Prevent Ethanol

Article pubs.acs.org/crt

Mitochondrial Permeability Transition Pore Inhibitors Prevent Ethanol-Induced Neuronal Death in Mice Frederic Lamarche,†,§,∥ Carole Carcenac,‡,§ Brigitte Gonthier,†,§,∥ Cecile Cottet-Rousselle,† Christiane Chauvin,† Luc Barret,†,§,∥ Xavier Leverve,†,§,∥,⊥ Marc Savasta,‡,§,# and Eric Fontaine*,†,§,∥,# †

Inserm, U1055, Laboratoire de Bioénergétique Fondamentale et Appliquée (LBFA) et SFR Biologie Environnementale et Systémique (BEeSy), Grenoble, F-38041, France ‡ Inserm, U836, Laboratoire Dynamique et Physiopathologie des Ganglions de la Base, Grenoble, F-38041, France § Joseph Fourier University, Grenoble, F-38041, France ∥ Grenoble University Hospital, Grenoble, F-38043, France S Supporting Information *

ABSTRACT: Ethanol induces brain injury by a mechanism that remains partly unknown. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). Here, we tested the effect of ethanol and PTP inhibitors on mitochondrial physiology and cell viability both in vitro and in vivo. Direct addition of ethanol up to 100 mM on isolated mouse brain mitochondria slightly decreased oxygen consumption but did not affect PTP regulation. In comparison, when isolated from ethanoltreated (two doses of 2 g/kg, 2 h apart) 7-day-old mouse pups, brain mitochondria displayed a transient decrease in oxygen consumption but no change in PTP regulation or H2O2 production. Conversely, exposure of primary cultured astrocytes and neurons to 20 mM ethanol for 3 days led to a transient PTP opening in astrocytes without affecting cell viability and to a permanent PTP opening in 10 to 20% neurons with the same percentage of cell death. Ethanoltreated mouse pups displayed a widespread caspase-3 activation in neurons but not in astrocytes and dramatic behavioral alterations. Interestingly, two different PTP inhibitors (namely, cyclosporin A and nortriptyline) prevented both ethanol-induced neuronal death in vivo and ethanol-induced behavioral modifications. We conclude that PTP opening is involved in ethanolinduced neurotoxicity in the mouse.

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prolonged PT leads to cell death,7 while the inhibition of PT prevents oxidative stress-induced cell death,8−10 including ischemia-reperfusion injury in humans.8−11 Ethanol intoxication leads to cell damage both in vivo and in isolated cells. In the brain, cell death is preceded by an increase in reactive oxygen species (ROS) production, a release of cytochrome c and AIF, and a decrease in mitochondrial ATP content and ATP synthesis.12−17 However, no studies have either checked if PTP inhibitors hampered ethanol toxicity in the brain or visualized PT in isolated brain cells. It has recently been reported that PTP inhibitor cyclosporin A (CsA) attenuates ethanol withdrawal-induced cell death in the HT22 cultured hippocampal cell line,18 while PTP opening has indisputably been shown to play a role in ethanol-induced toxicity in liver.19,20 In this work, we have explored the effects of ethanol in primary cultured neurons and astrocytes, as well as in a wellestablished model of fetal alcohol syndrome.21 Our results

INTRODUCTION Mitochondria are involved in several physiological processes including energy metabolism, calcium homeostasis, and programmed cell death. Indeed, in response to various stresses, apoptotic factors (such as cytochrome c and apoptosis inducing factor (AIF)) are released from the intermembrane space to the cytosol, which allows the execution of cell death programs.1−3 Mitochondrial permeability transition (PT) consists of a sudden increase in the permeability of the inner mitochondrial membrane, which becomes nonselectively permeable to molecules smaller than 1.5 kDa.4 This results in a marked inhibition of ATP synthesis through the collapse of the mitochondrial membrane potential and in the release of mitochondrial NADH.5 PT is believed to occur due to the opening of an inner membrane channel regulated by other mitochondrial proteins. This multiprotein complex is referred to as the permeability transition pore (PTP).4,6 Despite numerous efforts, the exact molecular nature of PTP remains elusive. Although the mechanism by which PTP opening triggers the release of mitochondrial pro-apoptotic mediators into the cytosol remains debated, it is well acknowledged that a © 2012 American Chemical Society

Received: September 19, 2012 Published: December 26, 2012 78

dx.doi.org/10.1021/tx300395w | Chem. Res. Toxicol. 2013, 26, 78−88

Chemical Research in Toxicology

Article

poly-D-lysine (50 μg/mL) and laminin (1 μg/mL). Cultures were seeded at a final density of 105 cells/cm2 and were maintained at a constant temperature of 37 °C in a humidified atmosphere of 5% CO2/95% air. Three days after plating, 50% of the culture medium was removed and replaced with fresh supplemented Neurobasal medium. Cells were used for experiments at 6 to 7 DIV. The growth, density, and morphologic characteristics of the cells were regularly observed by phase-contrast microscopy. Culture purity was estimated by immunocytochemical localization of neuron-specific enolase for neurons and of glial fibrillary acidic protein for astrocytes.25 Immunostaining showed that preparations obtained in the conditions described above contained more than 95% neurons. Ethanol Intoxication. Two different models, both of which led to ethanol-induced cell death, were used: first, an animal model of acute in vivo intoxication that reached a peak concentration of approximately 4g/L ethanol, followed by a continuous decrease due to animal ethanol metabolism, and second, an in vitro model in which cells were exposed to a long lasting and constant concentration of ethanol. For in vivo ethanol intoxication, seven-day-old C57BL/6 mice (from the same litter) were injected twice subcutaneously with 2 g/kg ethanol (20% in sterile saline) 2 h apart. Control pups were treated with saline solution only. This procedure has been shown to trigger neurodegeneration.26 The blood ethanol concentration of pups measured by GC/FID/HS (gas chromatography/flame ionization detector/head space) was 4 g/L and 0 g/L, 1 and 24 h after the second dose of ethanol, respectively. Thirty minutes before the first ethanol or saline injection, animals were treated subcutaneously with 20 mg/kg CsA, 2 mg/kg nortriptyline or vehicle, concentrations that have previously been shown to significantly reduce lesion volume27 and infarct size28 in rodent models of traumatic brain and ischemia reperfusion injuries, respectively. For in vitro ethanol intoxication, neurons (at 6 DIV) and astrocytes (at 14 DIV) were exposed to 20 mM ethanol for 3 days. In order to avoid ethanol evaporation during this incubation period, the culture dishes were incubated under an ethanol-saturated atmosphere, as described by Eysseric et al.29 CsA, nortriptyline, or vehicle was added 30 min before ethanol addition and remained in the culture medium through the whole experiment. Mitochondria Isolation, Mitochondrial Oxygen Consumption, ROS Production, and Permeability Transition Determination. After the removal of olfactory bulbs and the cerebellum, mouse brain mitochondria were isolated by standard differential centrifugation procedure in a medium containing 75 mM sucrose, 225 mM mannitol, 10 mM Hepes, and 1 mM EGTA (pH 7.4).30 Protein concentrations were determined in the bicinchoninic assay, using BSA as the standard (Pierce). Mitochondrial oxygen consumption rate (JO2) was measured polarographically at 30 °C. ROS production and Ca2+ measurements were performed fluorimetrically with a PTI Quantamaster C61 spectrofluorometer equipped with magnetic stirring and thermostatic controls. ROS production was assessed in the presence of 5 IU/mL horseradish peroxidase by monitoring H2O2induced fluorescence of 1 μM Amplex Red with excitation and emission wavelengths set at 560 and 584 nm, respectively.31 Calibration of H2O2 production was obtained by the addition of a known amount of H2O2. Extra-mitochondrial Ca2+ was measured in the presence of 1 μM calcium green-5N with excitation and emission wavelengths set at 506 and 530 nm, respectively. Ca2+ uptake and Ca2+ release of mitochondria were measured by loading mitochondria with trains of Ca2+ pulses at constant time intervals.32 Determination of Mitochondrial Area, Mitochondrial Membrane Potential and NAD(P)H Distribution by Confocal Microscopy. Cells (astrocytes or neurons) set on glass coverslips were studied by laser confocal microscopy at 37 °C in a humidified atmosphere (95% air, 5% CO2) using a microscope equipped with a perfusion chamber (POC chamber, LaCom, Erbach, Germany) and an incubation system (O2−CO2-°C, PeCom, Erbach, Germany). Images were collected with a Leica TCS SP2 AOBS inverted laser scanning confocal microscope equipped with a Coherent 351−364 UV laser using a 63× oil immersion objective (HCX PL APO 63.0 × 1.20 oil). Laser excitation was 351−364 nm for NAD(P)H, 488 nm for

demonstrate that ethanol leads to permanent PTP opening in primary cultured neurons, while PTP inhibitors prevent both neuronal death induced by ethanol in vivo and the subsequent modifications of animal behavior.

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MATERIALS AND METHODS

Animals. Pregnant C57BL6 mice were obtained from Charles River (L’Arbresle, France). All animal experiments were performed in compliance with the standards for animal care and housing of the guidelines of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS No. 123, Strasbourg, 18.III.1986). The Ethics Committee of Grenoble (ComEth) approved of this study and assigned it number 35 LBFA-FL-02. Reagents. Tetramethyl rhodamine methyl ester (TMRM), Mitotracker green, calcium green 5N, and anti-IgG coupled to Alexa 488 were purchased from Molecular Probes (Eugene, OR). Poly-Llysine, poly-D-lysine, laminin, adenosine diphosphate (ADP), oligomycin, 1-β-D-arabino-furanosyl-cytosine (AraC), nortripyline, calcium chloride, glutamate, malate, succinate, 3-[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT), mouse anti-GFAP, and DPX mounting medium were obtained from Sigma. DMEM/F12, neurobasal medium, B27 supplement, penicillin, streptomycin, and amphotericin B were purchased from Life Technologies. CsA was purchased from Novartis Pharma (France). Antiactive caspase-3 antiserum and anti-GFAP antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-IgG antibodies coupled to cyanin-3 were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Anti-NeuN antibodies were obtained from Millipore (Temacula, CA), while avidin−biotin-peroxidase conjugate (Vectastain ABC Elite) came from Vector Laboratories (Burlingame, CA). Other reagents were obtained from standard suppliers. Primary Astrocyte Cultures. Primary astrocyte cultures were prepared aseptically from the cerebral hemispheres of three- to fourday-old mice pups.22 Pups were killed by decapitation under sterile conditions. Cerebral hemispheres were dissected free of the meninx and gently dispersed in DMEM/F12 supplemented with 10% inactivated fetal calf serum and 1% penicillin, streptomycin, and amphotericin B. The resulting cell suspension was centrifuged at 180g for 5 min. The pellet was redispersed in the same serum-supplemented medium and filtered successively through cell strainers with 100 and 70 μm pores. The final concentration of the cell suspension was adjusted to 6 × 105 cells per milliliter of medium. The suspension was transferred into Petri dishes or glass coverslips coated with poly-Llysine (10 μg/mL). Cells were maintained at a constant temperature of 37 °C in a humidified atmosphere containing 5% CO2/95% air. Twenty-four hours after seeding, the culture medium was removed and replaced with fresh medium. The medium was changed every three days. The growth, density, and morphologic characteristics of the cells were observed regularly by phase-contrast microscopy. After 10 to 12 days in culture, the cells were shaken in order to eliminate microglia and oligodendrocytes. After 14 days in vitro (14 DIV), the monolayer consisted of 98% astrocytes, as demonstrated by positive immunostaining with an antiserum against a-GFAP, a marker of astrocytes.23 Primary Neuron Cultures. Neuron cultures were obtained from 17-day-old mouse embryos by a modified version of the method described by Daval et al.24 Pregnant C57BL/6 mice were killed by cervical dislocation. Living embryos were excised by cesarean section under sterile conditions and killed by decapitation. Cerebral hemispheres were dissected free of the meninx in a HBSS medium supplemented with D-glucose and Hepes, with no antibiotics. A cell suspension was obtained by incubating hemispheres in a 2.5% trypsin solution, followed by supplemented HBSS plus BSA (bovine serum albumin) and DNase. The cell suspension was then centrifuged at 280g for 5 min. The pellet was redispersed in Neurobasal medium supplemented with B27-supplement and pyruvate, and filtered through a fine nylon mesh (40 μm pores). The final density of the cell suspension was adjusted to 106 cells/mL. The suspension was transferred into culture dishes or glass coverslips precoated with 79



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Table 1. Effect of Ethanol on Mitochondrial Oxygen Consumption and H2O2 Productiona ROS production (pmol H2O2·min−1·mg prot−1)

respiration (nmol O2·min−1·mg prot−1) glutamate/malate state 3 control control + 100 mM EtOH EtOH 1 h EtOH 24 h

135.2 ± 5.2 108.8 ± 4.2** 86.2 ± 6.6*** 138.2 ± 9.6

succinate

state 4

state 3

± ± ± ±

120.0 ± 6.6 95.4 ± 3.0* 78.8 ± 7.8** 115.4 ± 10.4

16.6 20.2 13.0 18.6

1.0 1.4 1.0* 2.4

state 4 32.6 35.0 21.0 32.8

± ± ± ±

1.2 1.6 2.0*** 4.8

glutamate/malate 1599 1683 1749 1371

± ± ± ±

107 165 179 119

succinate 13707 12423 13198 12773

± ± ± ±

1344 2378 2234 686

The incubation medium contained 125 mM KCl, 20 mM Tris, 5 mM Pi, and 1 mM EGTA. The final volume was 1 mL (pH 7.20) at 30 °C. Experiments were started by the addition of 0.5 mg mitochondria isolated from control or intoxicated animals (1 or 24 h after in vivo administration). When indicated, control mitochondria were studied in the presence of 100 mM ethanol. Oxygen consumption rate was measured in the presence of either 5 mM glutamate and 2.5 mM malate, or 5 mM succinate. State 3 and state 4 were measured after the addition of 500 μM ADP and 0.50 μg oligomycin/mg of protein, respectively. H2O2 production was measured in the presence of either 5 mM glutamate and 2.5 mM malate, or 5 mM succinate in a medium supplemented with 5 UI/mL horseradish peroxidase and 1 μM Amplex Red. Results are the mean ± SE of at least 4 different experiments. Data were compared by analysis of variance (ANOVA) and means by the Fisher PLSD test (Statview). *p < 0.05, ** p < 0.01, and ***p < 0.001 versus control. a

Mitotracker green and 543 nm for TMRM. The fluorescence emission wavelength, adjusted with AOBS, was 390−486 nm for NAD(P)H, 510−530 nm for Mitotracker green, and 565−645 nm for TMRM. In order to allow the overlay of NAD(P)H and TMRM or Mitotrackergreen signals, image acquisition was set with the same pinhole aperture (Airy 2.25), necessarily increased because of the low signal of NAD(P) H autofluorescence. Image quantification was performed using ImageJ (NIH images) and Volocity (Improvision) software as described by Dumas et al.5 Background noise of NAD(P)H autofluorescence was removed by a fine filter (Kernel 3 × 3) using Volocity software, while the images of TMRM and Mitotracker green were not electronically manipulated. MTT Assay. Cell viability was assessed spectrophotometrically by measuring the reduction of 3-[4, 5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide to formazan.33 Neurons or astrocytes were incubated for 3 h at 37 °C with MTT (500 μg/mL), washed with icecold phosphate-buffered saline, and lysed in dimethyl sulfoxide. Absorbance was measured at 570 nm with an Amersham Biosciences Ultrospec 2100 pro. For an easier comparison, data were normalized (i.e., divided by the proper control). Immunohistochemical Studies. Six hours after the second ethanol or saline injection, the pups were deeply anesthetized with an intraperitoneal injection of 0.1% pentobarbital. Brains were quickly removed and immersed overnight in a fixative solution containing 4% paraformaldehyde in phosphate buffer. Brains were then immerged in 20% sucrose in 0.1 phosphate buffer, pH 7.4, frozen in cooled (−40 °C) isopentane, and then stored at −30 °C. Serial frontal sections (30 μm) were cut on a Microm HM 500 cryostat and processed for caspase-3, NeuN, and GFAP immunohistochemistry. Caspase-3 Positive Cells. Free-floating sections were thoroughly washed with PBS, and endogenous peroxidase activity was blocked by incubation in a solution of methanol containing 3% hydrogen peroxide, and then incubated for 1 h in blocking solution (2% BSA/ 0.2% milk/0.1% Triton-X 100 in PBS), followed by an overnight incubation in rabbit antiactive caspase-3 antiserum diluted 1:1500 in blocking solution at 4 °C. Immunoreactivity was detected with avidin− biotin-peroxidase conjugate for 1 h, using 3,3′-diaminobenzidine as the substrate for color reaction. Sections were then washed 3 times with PBS and were then dehydrated by incubation in a series of graded ethanol solutions, cleared in xylene, and coverslipped with DPX mounting medium for microscopy and quantification. The number of caspase-3 immunoreactive cells was evaluated under a motorized Nikon Eclipse microscope equipped with a high-resolution digital camera coupled with a computerized image analysis system using the ICS Framework program. For each animal, the number of caspase-3 immunoreactive cells was counted on three coronal brain sections for each region of interest. Cell numbers were expressed as the mean number/section.

Co-Labeling Caspase-3/NeuN and Caspase-3/GFAP. Freefloating sections were thoroughly washed with PBS and incubated for 1 h in 2% BSA/0.2% milk/0.1% Triton-X 100 in PBS. Sections were then incubated for 24 h at 4 °C in rabbit antiactive caspase-3 antiserum diluted 1:1500 and mouse anti-NeuN diluted 1:500 or mouse antiGFAP diluted 1:1000 in blocking solution. Immunoreactivity was detected by incubation with goat antirabbit IgG coupled with Alexa 488 and goat antimouse IgG coupled to Cyanin-3. Behavioral Tests. The spontaneous maternal odor preference test34 was carried out three days after ethanol treatment. Two glass boxes 2.5 cm away from each other and filled with litter (home litter on the one hand and clean litter on the other hand) were covered with a metallic mesh floor. The test was started by placing the pup in the neutral zone (i.e., on the mesh between the two glass boxes) and the experimenter measured the time spent over each glass box for 60 s (defined by the time the pup’s head crossed one of the edges of the neutral zone). Each pup underwent three successive tests. The direction the pup faced when placed in the neutral zone was changed (i.e., 180° rotation) at the beginning of each test. The mesh and the neutral zone were wiped between each experiment. The nociception test35 was carried out a few days after weaning. As pain and temperature sensation follow the same neuroanatomical circuit (i.e., the anterolateral system), this circuit was studied by introducing the mice into an open-ended cylindrical space, with a floor consisting of a plate heated to a constant temperature of 56 °C. This temperature is unpleasant but well below the pain threshold. This procedure generated two behaviors: paw licking and jumping, for which reaction times could be measured. Each mouse underwent the test only once, as a learning phenomenon has been reported for this test. Except the time spent over each glass box, and the time before paw licking and jumping, no other physiological parameters were monitored. Therefore, these two behavioral tests were not expected to induce suffering or anxiety.

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RESULTS Effect of in Vitro and in Vivo Alcohol Exposure on Mitochondrial Functions. We first checked whether ethanol affected mitochondrial functions in isolated mouse brain mitochondria. Mitochondria were isolated from control or ethanol-intoxicated mice either 1 h (blood ethanol concentration = 4 g/L) or 24 h after the second ethanol injection (i.e., after blood ethanol concentration had returned to zero). As seen in Table 1, in vitro addition of 100 mM (approximately 4g/L) ethanol slightly decreased the oxygen consumption rate in state 3 (a condition where mitochondria consume oxygen mainly to sustain ATP synthesis) but not in state 4 (a condition where mitochondria are at rest and consume oxygen only to

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Figure 1. Effect of CsA, nortriptyline, and ethanol on the Ca2+ retention capacity of isolated mouse brain mitochondria. The incubation medium contained 250 mM sucrose, 1 mM Pi, 10 mM Tris-MOPS, 5 mM glutamate, 2.5 mM malate, 1 μM calcium green-5N. The final volume was 1 mL (pH 7.40) at 25 °C. Experiments were started by the addition of 0.5 mg of mitochondria. Where indicated, 25 μM Ca2+ pulses were added (arrows). In panels A−D, mitochondria were incubated in the absence (A) or presence of 1 μM CsA (B), 2.5 μM nortriptyline (C), and 100 mM ethanol (D). In panels E and F, brain mitochondria were isolated from an ethanol-treated mouse 1 h (E) or 24 h (F) after in vivo ethanol administration. Results are representative of at least 6 different experiments. The CRC in the absence of inhibitor (control) was 171 ± 2.3 μmol/mg mitochondria, whereas it was 240 ± 3.7 and 217 ± 4.4 in the presence of CsA and nortriptyline, respectively. Results are the mean ± SE of 6 different experiments; p < 0.05, between control and CsA or nortriptyline, using the paired Student’s t test.

Effect of Ethanol Exposure on Primary Cultured Astrocytes and Neurons. Primary cultured astrocytes (Figure 2) and primary cultured neurons (Figure 3) were studied by confocal microscopy after loading with Mitotracker (which colocalizes with mitochondria37) and TMRM (which accumulates in mitochondria according to the mitochondrial membrane potential38). In normal conditions, NAD(P)H is mainly found inside mitochondria. As a consequence, NAD(P)H autofluorescence colocalizes with both Mitotracker (i.e., mitochondrial area) and TMRM fluorescence in control conditions.5 It has long been recognized that PT is a reversible event. However, not all the consequences of PT are reversible. The closure of PTP restores the mitochondrial membrane potential and the ionic homeostasis of compounds that are physiologically transported, whereas NAD(P)H (which only diffuses through an open PTP) remains excluded after it has left the mitochondria. Putting this feature into practice, we have shown that permanent PTP opening leads to mitochondrial depolarization (i.e., a decrease in TMRM signal) and a release of NAD(P)H outside mitochondria, leading to an increase in NAD(P)H area/Mitotracker area ratio (area being defined as the sum of the pixel above a threshold value) as well as a decrease in TMRM fluorescence/Mitotracker fluorescence ratio (fluorescence being defined of the sum of the fluorescence of the pixel of the area).5,39 Conversely, in the case of transient PTP opening, the NAD(P)H released outside mitochondria during PTP opening remains in the cytosol after PTP closing because there is no specific channel for NAD(P)H exchange (i.e., for NAD(P)H uptake), whereas the proton leak through the opened PTP stops after PTP closing, resulting in a normal membrane potential. This leads to an increase in the NAD(P)H area/Mitotracker area ratio, whereas the TMRM fluorescence/ Mitotracker fluorescence ratio remains unchanged.5 As seen in Figure 2B, NAD(P)H can easily be seen outside mitochondria in several astrocytes when exposed to 20 mM

maintain the mitochondrial membrane potential). The effect of ethanol administered in vivo was stronger, decreasing the oxygen consumption rate in both resting and phosphorylating conditions. However, this effect was transient and disappeared after ethanol had been eliminated (i.e., after 24 h). Note that neither in vitro nor in vivo ethanol exposure affected H2O2 production in isolated brain mitochondria. We next checked whether ethanol affected PTP regulation. Ca2+ is the single most important factor for PTP opening. Ca2+ retention capacity (CRC) represents the minimum Ca2+ load required to induce PTP opening in an entire population of mitochondria. PTP-inhibitors and so-called PTP-inducers refer to factors that increase and decrease the amount of Ca2+ required to induce PTP opening (i.e., CRC).36 As seen in Figure 1, neither in vitro nor in vivo exposure to ethanol affected PTP regulation in isolated brain mitochondria. Note, however that, as expected, the reference pharmacological PTP inhibitor cyclosporin A (CsA) increased CRC (i.e., inhibited PTP opening). Moreover, nortriptyline, which has been reported to inhibit PTP opening in rat brain mitochondria28 also increased CRC in mouse brain mitochondria. In summary, the presence of ethanol decreased brain mitochondrial respiration transiently without increasing H2O2 production or sensitizing mitochondria to calcium. These results highlight the complexity and subtlety of the effects of ethanol on brain mitochondrial physiology. Nevertheless, it has been shown that ethanol induces cell death in mouse brain at the concentration used here,21 albeit in a small percentage of cells only (see below). In other words, mitochondria were isolated mainly from cells that had not undergone apoptosis. Moreover, mitochondria isolation procedures remove cytosolic signals that may affect mitochondria in vivo. To skirt these issues, we next studied the occurrence of PTP opening and cell death directly in intact cells. 81



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Figure 2. Effect of 3-day ethanol exposure on PTP opening and cell death in cultured astrocytes. (A,B) Triple channel imaging of mitochondrial area, mitochondrial electrical membrane potential, and NAD(P)H autofluorescence. Primary mouse cultured astrocytes exposed or not to 20 mM ethanol for 3 days were loaded with 50 nM Mitotracker green (MTG) and 10 nM TMRM. The fluorescence of MTG (green), TMRM (red), and NAD(P)H (blue) was imaged simultaneously by confocal microscopy. Image bar, 47 μm. (C,D) Fluorescence quantification. MTG, TMRM, and NAD(P)H quantification was calculated with ImageJ and Volocity software. Area represents the sum of all the pixels above a threshold value determined by ImageJ. Fluorescence represents the whole fluorescence of the pixels within the area. Results are the mean ± SE of at least 15 randomly chosen fields from 4 different experiments. Unpaired Student’s t test was used. (E) Cell viability. Primary mouse cultured astrocytes exposed or not to 20 mM ethanol for 3 days in the presence or absence of either 1 μM CsA or 2.5 μM nortriptyline. Cell viability was assessed by MTT assay as described in Materials and Methods. Results are the mean ± SE of 15 assays from at least 3 different experiments.

spared astrocytes (Figure 2E). Confirming that ethanol-induced cell death was due to PTP opening, we discovered that two different PTP inhibitors (namely, CsA and nortriptyline) prevented ethanol-induced neuron death (Figure 3E). In Vivo Exposure of Mouse Pups to Ethanol Induces PTP Inhibitor-Sensitive Caspase-3 Activation in Neurons but Not in Astrocytes. In order to check whether ethanol brain toxicity was due to PTP opening in vivo, we next studied the effects of CsA and nortriptyline on a well-characterized animal model of ethanol-induced brain injury. As expected,21 immunohistochemical staining with antibodies against activated caspase-3 carried out 6 h after subcutaneous ethanol injections showed that ethanol induced a strong and significant caspase-3 activation in many areas of the brain (Figure 4A). Computerassisted analysis revealed that there was a spontaneous number of caspase-3 positive cells in control animals. However, as compared with controls, ethanol treatment increased the number of dying cells by a factor of 20 in the piriform cortex, 60 in the olfactory tubercles, 65 in the cingulate cortex, 90 in the somatosensory cortex, 100 in the motor cortex, 150 in the hippocampus, and 400 in the thalamus (Figure 4B). As shown in Figure 4, a single subcutaneous injection of either CsA (20

ethanol for 3 days, leading to an increase in the NAD(P)H area/Mitotracker area ratio as compared with control astrocytes (Figure 2C). On the contrary, ethanol did not affect the TMRM fluorescence/Mitotracker fluorescence ratio (i.e., mitochondrial membrane potential) in primary cultured astrocytes (Figure 2D), suggesting that ethanol exposure led to transient PTP opening in astrocytes. Interestingly, the same ethanol exposure led to a different response when applied to primary cultured neurons. As exemplified in Figure 3B, 10 to 20% neurons exposed to ethanol displayed an increase in the NAD(P)H area/Mitotracker area ratio (Figure 3C) and a decrease in the TMRM fluorescence/Mitotracker fluorescence ratio (Figure 3D), suggesting that ethanol exposure led to permanent PTP opening in some neurons. Note that in the absence of ethanol (Figure 3A) or in the presence of ethanol and CsA or nortriptyline (see Supporting Information), we did not observe neurons with an increase in the NAD(P)H area/ Mitotracker area ratio or a decrease in the TMRM fluorescence/Mitotracker fluorescence ratio. In agreement with the observation that permanent (but not transient) PTP opening leads to cell death,40 ethanol induced cell death in approximately 20% neurons (Figure 3E) but 82



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Figure 3. Effect of 3-day ethanol exposure on PTP opening and cell death in cultured neurons. (A,B) Triple channel imaging of mitochondrial area, mitochondrial electrical membrane potential, and NAD(P)H autofluorescence. Primary mouse cultured neurons exposed or not to 20 mM ethanol for 3 days were loaded with 50 nM Mitotracker green (MTG) and 10 nM TMRM. The fluorescence of MTG (green), TMRM (red), and NAD(P)H (blue) was imaged simultaneously by confocal microscopy. Image bar, 20 μm. The result in panel A (control) is representative of all the randomly chosen fields. The result in panel B (ethanol) is representative of 10 to 20% of randomly chosen fields (80 to 90% of the fields were similar to those in panel A). (C,D) Fluorescence quantification. MTG, TMRM, and NAD(P)H quantification was calculated with ImageJ and Volocity software. Area represents the sum of all the pixels above a threshold value determined by ImageJ. Fluorescence represents the whole fluorescence of the pixels within the area. Results are the mean ± SE of at least 11 fields (randomly chosen fields for the control and representative for ethanol treatment) from 4 different experiments. Unpaired Student’s t test was used. (E) Cell viability. Primary mouse cultured neurons exposed or not to 20 mM ethanol for 3 days in the presence or absence of either 1 μM CsA or 2.5 μM nortriptyline. Cell viability was assessed by the MTT assay as described in Materials and Methods. Results are the mean ± SE of 15 assays from 3 different experiments. Data were compared by an analysis of variance (ANOVA) and means by the Fisher PLSD test (Statview).

mg/kg) or nortriptyline (2 mg/kg) 30 min before ethanol treatment dramatically decreased the number of caspase-3activated cells, strongly suggesting that caspase-3 activation was due to PTP opening. Note that CsA (20 mg/kg) or nortriptyline (2 mg/kg) did not affect the spontaneous number of caspase-3 positive cells in animals that were not exposed to ethanol (see Supporting Information). In order to specify the type of cells killed by ethanol in vivo, we next coupled activated caspase-3 immunostaining with the use of NeuN or GFAP antibodies, specific to neurons and astrocytes, respectively. As seen in Figure 5, confocal microscopy showed that activated caspase-3 fluorescence (green) colocalized with NeuN fluorescence but not with GFAP fluorescence. This result suggests that neurons are more vulnerable than astrocytes to the apoptogenic effect of ethanol in our animal model. In Vivo Exposure of Mouse Pups to Ethanol Induces PTP Inhibitors-Sensitive Behavioral Modifications. We finally determined whether the observed neuronal loss was associated with cognitive dysfunctions and somatosensory impairments, and whether the neuroprotective effects of CsA

and nortriptyline prevented such behavioral alterations. Two sensorimotor tests suitable for newborn or young mice were performed. First, the spontaneous preference for maternal odor, as an index of odor memory,34 was performed on postnatal day 10. Second, the nociceptive response to heat was performed a few days after weaning by dropping mice onto a heated metal plate.35 As expected, control animals remained significantly longer over their home litter than the clean litter (Figure 6A). On the contrary, ethanol-exposed animals spent the same time over the two litters. In the nociceptive test (Figure 6B), the time before the first jump was longer among ethanol-exposed animals as compared with controls. Suggesting that these behavioral impairments were directly related to ethanol-induced PTP opening, CsA and nortiptyline improved the behavior of ethanol-exposed animals (Figure 6).

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DISCUSSION In this work, we have used two different models that both lead to ethanol-induced cell death. The animal model faithfully reproduces some major neuropathological aspects of the human 83



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Figure 4. CsA and nortriptyline protect mouse brain from ethanol-induced caspase-3 activation. Subcutaneous injection of CsA, nortriptyline, or vehicle, followed by 2 subcutaneous injections of ethanol or saline, was performed in 7-day-old C57BL6 mice as indicated in Materials and Methods. Brains were removed 6 h after the second ethanol injection and fixed for subsequent staining by an antibody against activated caspase-3. Quantification of caspase activated positive cells was performed by computerized image analysis. CgCx = cingulate cortex, MCx = motor cortex, PirCx = piriform cortex, SSCx = somatosensory Cortex, Hippo = hippocampus, and TU = olfactory tubercles. Results are the mean ± SE of at least 8 animals. Data were compared by an analysis of variance (ANOVA) and means by the Fisher PLSD test (Statview).

fetal alcohol syndrome, while the in vitro model allows the direct visualization of PT in isolated cells. Whatever the model used, we have shown that the inhibition of PT prevents ethanol-induced neuron death. To the best of our knowledge, this is the first demonstration that PTP opening is involved in ethanol-induced brain injury. CsA is the reference PTP inhibitor but is also known to inhibit calcineurins. Whether CsA prevents cell death because it inhibits PT or calcineurins is usually confirmed either by using another PTP inhibitor with no effect on calcineurins (such as

nortriptyline) or using a calcineurin inhibitor that does not affect the PTP. In this work, we have used the first strategy. Moreover, the question of whether CsA prevents cell death because it inhibits PT or because of some other mechanisms has been definitively solved by the demonstration that genetic ablation of cyclophilin D (the molecular target of CsA) inhibits PT and prevents cell death just as CsA does including in models of brain damages. 10 Because PTP opening in intact cells required the measurement of NAD(P)H (Figures 2 and 3) while ethanol metabolism 84



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Figure 5. Ethanol induces caspase-3 activation in neurons. Animals were treated as described in Figure 4. Brains were removed 6 h after the second ethanol injection and fixed for subsequent staining by antibodies against activated caspase-3 (green) and anti-GFAP (red, panel A) or anti-NeuN (red, panels B and C). Images are representative of 5 different experiments. Image bar, 300 μm.

increases CypD acetylation, which may promote its binding to the PTP.44 Such an effect has been shown to sensitize PTP opening in H4IIEC3 cells.44 Note, however, that ethanol exposure did not favor PT in isolated brain mitochondria (Figure 1). This observation does not rule out an increase in CypD binding to the PTP in cells that will undergo apoptosis but does not support a general increase in CypD binding to the PTP in all the cells exposed to ethanol. It is now well acknowledged that PTP regulation is partly tissue-specific. Indeed, depending on the cells studied, the amount of Ca2+ required to induce PT may change dramatically, while several drugs may regulate PTP opening differently in various tissues.32,45,46 From a theoretical point of view, it is therefore expected that the same stress opens the PTP (and induces cell death) in certain types of cells, while sparing others. In this work, we report that the same ethanol exposure induced transient (Figure 3) or permanent (Figure 4) PTP opening in astrocytes or neurons, respectively, suggesting that PTP is more prone to open in neurons than in astrocytes. In various models of brain injury, astrocytes have been found to be more resistant than neurons. It has been suggested that such differences may be due to higher antioxidant defenses in astrocytes than in neurons.47−49 However, this does not rule out the proposal that PTP opening might be easier in neurons than in astrocytes because it is well known that reactive oxygen species favor PTP opening.4 Assuming that spontaneous cell death in control animals is the same regardless of the brain area, the fact that caspase activation (caspase positive cell after ethanol/caspase positive

produces NADH, one may question the results of these experiments. However, catalase and CYP2E1 (which do not produce NADH) are the key enzymes of ethanol oxidation in the brain of rodents, whereas alcohol dehydrogenase (which produces cytosolic NADH) plays a minor role, if any, in this tissue.41 Moreover, acetaldehyde is converted to acetic acid and NADH inside mitochondria. However, such a mitochondrial production cannot account for the observed increase in cytosolic NADH (in the absence of PTP opening). Currently, the mechanism by which ethanol triggers PTP opening remains unknown. Experiments using isolated mitochondria suggest that ethanol per se does not induce PTP opening, neither when ethanol is directly added to mitochondria nor when mitochondria are isolated from animals exposed to ethanol. Nevertheless, the conclusion that ethanol leads to PTP opening is sustained by the direct observation of PT in neurons (Figure 3) and the prevention of cell death using two different PTP inhibitors (Figures 3 and 4). It is therefore highly probable that ethanol indirectly induces PTP opening either via a signaling process or via the direct toxicity of one of its metabolites. Ethanol has been reported to induce oxidative stress,42 which might trigger PTP opening.4,6,36 Note, however, that ethanol did not increase mitochondrial H2O2 production in our animal model after mitochondria had been isolated (Table 1). The matrix protein cyclophilin D (CypD) is the best-defined regulatory component of PTP.8−10,43 Although the molecular target of CypD on the PTP has not been found yet, the occurrence of PT is made easier when CypD binds to the PTP.43 It has recently been reported that ethanol exposure 85



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Confirming that these behavioral impairments were directly related to PTP opening, CsA and nortiptyline improved the behavior of ethanol-exposed animals (Figure 6). Note that the olfactory test studied several brain areas in which CsA was less potent than nortiptyline (Figure 4). Consequently, CsA partly normalized this test, while nortiptyline did it totally. Although CsA was as potent as nortriptyline at PTP inhibition in isolated brain mitochondria (Figure 1) and at preventing ethanol-induced cell death in isolated neurons (Figure 3E), nortriptyline was more efficient than CsA at brain protection, especially in the cingulated, motor, and piriform cortices, and the olfactory tubercles (Figure 4B). Whether CsA crosses the intact blood−brain barrier in sufficiently large amounts to inhibit PTP opening and to promote cerebral cell survival has been questioned.55 However, the young animals used in this work are known to have a developing blood−brain barrier, which is more permeable than that of fully developed animals, especially for CsA.56 The protective effect of CsA in brain (Figure 4) confirmed that CsA reached its target in our model. However, depending on the brain area considered, the final concentration of CsA may vary, accounting for the observed differences in brain protection. Acute and chronic alcohol-induced brain injuries are true public health problems. The animal model used in this work21 suggests that fetal alcohol symptoms can be attenuated by PT inhibition. We did not demonstrate here whether brain damage induced by chronic alcohol abuse in adults involved PTP opening, but this hypothesis seems likely. Of course, the best way to protect the brain from ethanol injury is obviously not to use PTP inhibitors but to refrain from drinking alcohol in the first place. However, this work provides new perspectives for the prevention of fetal alcohol spectrum disorder in children whose mothers drank alcohol during pregnancy and for the prevention of ethanol-induced brain injuries in cases of isolated and acute alcohol consumption (i.e., binge drinking). It could also facilitate the implementation of strategies for preventing and/or decreasing the appearance and progression of other neurodegenerative diseases in which PTP opening might occur.

Figure 6. CsA and nortriptyline protect mouse from ethanol-induced behavioral impairment. Animals were treated by ethanol in the presence of CsA, nortriptyline, or vehicle as in Figure 4. The spontaneous maternal odor preference test (panel A) and the hot plate test (panel B) were performed 3 and 19 days after ethanol intoxication, respectively. Results are the mean ± SE of at least 13 animals per condition. Data were compared by analysis of variance (ANOVA) and means by the Fisher PLSD test (Statview). *p < 0.01, ** p < 0.001, ***p < 0.0001, significantly different from the respective control. $p < 0.05, $$$p < 0.0001, significantly different from the respective EtOH.

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cell in the control) dramatically differed from one area to the other (see Figure 4) suggests that the sensitivity of neurons may depend on their localization. As ethanol indirectly induces PTP opening while ethanol is expected to distribute homogeneously into the brain, two mutually nonexclusive hypotheses can be proposed: either ethanol-induced cell signaling and/or ethanol metabolism may change according to neuron location or PTP may be intrinsically regulated differently according to neuron location. In agreement with previous results showing a correlation between ethanol-induced brain neurodegeneration and spatial learning and memory impairments in juvenile mice,50 ethanol intoxication affected spontaneous maternal odor preference in 10-day-old mice and the nociceptive response in 26-day-old mice (Figure 6). Note that these tests were performed 2 and 18 days after the blood ethanol concentration had return to zero. Although we cannot exclude for good an effect due to ethanol withdrawal stress (especially for the spontaneous maternal odor preference test), we think this proposal unlikely using such an acute model. Indeed, these results are consistent with the brain areas displaying ethanol-induced lesions as the central olfactory tract involves the olfactory tubercles, piriform cortex, thalamus, and hippocampus, 51,52 while the pain circuit and the nociceptive response involve the thalamus as well as the motor and somatosensory cortices.53,54

ASSOCIATED CONTENT

S Supporting Information *

The effect of 3-day ethanol exposure on PTP opening and cell death in cultured neurons incubated in the presence of CsA or nortiptyline, as well as the effect of CsA and nortriptyline on caspase-3 activation of mouse brain from control animals. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

Co-last authors.

Funding

This work was supported by a grant from Institut National de la Santé et de la Recherche Médicale (Programme National de Recherche Alcool). Notes

The authors declare no competing financial interest. ⊥ Deceased November 8, 2010. 86



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ACKNOWLEDGMENTS We thank Cindy Tellier (our animal keeper) and Nathalie Signorini-Allibe (GC/FID/MS measurements) for their invaluable assistance. We also thank Christophe Cottet for the English corrections to this paper.

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ABBREVIATIONS AIF, apoptosis inducing factor; CRC, Ca2+ retention capacity; CsA, cyclosporin A; CypD, cyclophilin D; DIV, days in vitro; EtOH, ethanol; MTT, 3-[4, 5-dimethyl-thiazol-2-yl]-2, 5diphenyltetrazolium bromide; Norti, nortriptyline; Pi, inorganic phosphate; PT, permeability transition; PTP, permeability transition pore; ROS, reactive oxygen species; TMRM, tetramethyl rhodamine methyl ester

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Mitochondrial Permeability Transition Pore Inhibitors Prevent Ethanol

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