ORIGINAL RESEARCH ARTICLE
Cytosolic Zinc Accumulation Contributes to Excitotoxic Oligodendroglial Death Susana Mato,1,2,3 Marı´a Victoria S anchez-G omez,1,2,3 Ana Bernal-Chico,1,3 and Carlos Matute1,2,3 Dyshomeostasis of cytosolic Zn2þ is a critical mediator of neuronal damage during excitotoxicity. However, the role of this cation in oligodendrocyte pathophysiology is not well understood. The current study examined the contribution of Zn2þ deregulation to oligodendrocyte injury mediated by AMPA receptors. Oligodendrocytes loaded with the Zn2þ-selective indicator FluoZin-3 responded to mild stimulation of AMPA receptors with fast cytosolic Zn2þ rises that resulted from intracellular release, as they were not blocked by the extracellular Zn2þ chelator Ca-EDTA. Pharmacological experiments suggested that AMPA-induced Zn2þ mobilization depends on cytosolic Ca2þ accumulation, arises from mitochondria and protein-bound pools, and is triggered by mechanisms that do not involve the generation of reactive oxygen species. Moreover, intracellular Zn2þ rises resulting from AMPA receptor activation seem to be promoted by Ca2þ-dependent cytosolic acidification. Addition of the cell-permeable Zn2þ chelator TPEN significantly reduced mitochondrial membrane depolarization, reactive oxygen species production, and cell death by sub-maximal activation of AMPA receptors both in vitro and in situ, suggesting that Zn2þ deregulation is an important mediator of oligodendrocyte excitotoxicity. These data provide evidence that strategies aimed at maintaining Zn2þ homeostasis may be useful for the treatment of disorders in which excitotoxicity is an important trigger of oligodendroglial death. Key words: oligodendrocyte; AMPA receptor; excitotoxicity; acidosis; ROS; mitochondria; Zn2þ GLIA 2013;00:000–000
INTRODUCTION
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lutamate excitotoxicity is a key pathogenic mechanism of white matter damage during acute and chronic insults to the central nervous system, including ischemia, brain trauma, and multiple sclerosis. Oligodendrocytes express AMPA/Kainate and NMDA receptors (Patneau et al., 1994; Salter and Fern, 2005), and are susceptible to damage by excessive glutamate signaling both in vitro (Matute et al., 1997; McDonald et al., 1998; Rosenberg et al., 2003; SanchezGomez et al., 2011) and in vivo (Follett et al., 2000; Matute 1998). Indeed, ionotropic glutamate receptor antagonists ameliorate neurological deficits and prevent oligodendroglial death in animal models of cerebral ischemia and multiple sclerosis (Pitt et al., 2000; Salter and Fern, 2005; Tekk€ok and Goldberg, 2001). Excitotoxicity to oligodendrocytes is often
initiated by excessive Ca2þ entry through AMPA and kainate receptors, which triggers mitochondrial depolarization, generation of reactive oxygen species (ROS) (Sanchez-Gomez et al., 2003), and activation of caspase-dependent and -independent death mechanisms (Sanchez-Gomez et al., 2003). However, the steps leading to excitotoxic oligodendrocyte injury downstream of Ca2þ overload have not been fully elucidated. Zinc in its ionic form (Zn2þ) is released upon activity of many glutamatergic terminals and plays crucial roles in the pathophysiology of brain function. It is now well recognized that Zn2þ influx from the extracellular space and its mobilization from intracellular pools such as mitochondria, cytosolic Zn2þ binding proteins, and lysosomes trigger mitochondrial dysfunction and ROS production, leading to neuronal damage under a variety of conditions associated with
View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22470 Received Aug 2, 2012, Accepted for publication Dec 27, 2012. Address correspondence to Carlos Matute or Susana Mato, Department of Neurosciences, University of of the Basque Country-UPV/EHU, Leioa 48940, Bizkaia, Spain. E-mail:
[email protected] or
[email protected] ogico de Vizcaya, E-48170 Zamudio, From the 1Departamento de Neurociencias, Universidad del Paı´s Vasco-UPV/EHU, E-48940 Leioa, Spain; Neurotek, Parque Tecnol on Biom edica en Red en Enfermedades Neurodegenerativas (CIBERNED), Spain; 3Achucarro Basque Center for Neuroscience-UPV/EHU, Spain; 2Centro de Investigaci E-48170 Zamudio, Spain. Grant sponsor: MICINN, Grant number: SAF2010-21547; Grant sponsor: CIBERNED. Additional Supporting Information may be found in the online version of this article.
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excitotoxicity (Dineley et al., 2008; Kiedrowski 2011; Sensi et al., 2011). Excessive cytosolic Zn2þ depletion can also be deleterious to neurons (Sensi et al., 2011), suggesting that tight regulation of Zn2þ homeostasis is crucial for neuronal survival. Supporting this notion, neurons possess a complex machinery for cytosolic Zn2þ transport and buffering, including a multitude of Zn2þ transporters (ZnTs) that lower cytosolic Zn2þ levels by acting as Zn2þ/Hþ or Kþ antiporters, Zn2þ-importing proteins (ZIPs) that promote Zn2þ transport from the extracellular space or organellar lumen to the cytosol, and cytosolic Zn2þ-binding proteins of the metallothionein (MT) family (Cousins et al., 2006; Sensi et al., 2009). The molecular mechanisms responsible for Zn2þ homeostasis in oligodendrocytes remain largely unexplored. Oligodendrocyte precursors seem to possess a high-affinity Zn2þ uptake mechanism (Law et al., 2003), and anatomical studies suggest the expression of ZIP-1 and ZnT-1 in white matter tracts (Belloni-Olivi et al., 2009; Nitzan et al., 2002; Sekler et al., 2002). Supporting the physiopathological relevance of Zn2þ dyshomeostasis in oligodendrocytes, these cells are vulnerable to extracellular Zn2þ insults (Kelland et al., 2004) and to cytosolic Zn2þ rises resulting from intracellular release, as the cytotoxic effects of peroxynitrite, a powerful oxidant that contributes to white matter damage in multiple sclerosis and spinal cord injury (Li et al., 2011b; Xiong and Hall 2009), are initiated by Zn2þ liberation from cytosolic pools (Li et al., 2011a; Zhang et al., 2007). Here we investigated the potential contribution of Zn2þ dyshomeostasis to oligodendrocyte excitotoxicity. Activation of oligodendrocyte AMPA receptors triggered rapid elevations in the levels of cytosolic Zn2þ, which resulted from intracellular mobilization of the metal. Mechanistic analysis indicated that Ca2þ-dependent cytosolic acidification is the main mediator of AMPA-induced Zn2þ deregulation in oligodendrocytes. Zn2þ accumulation contributed to mitochondrial dysfunction, ROS production, and oligodendrocyte excitotoxicity mediated by AMPA receptors. Thus, interventions targeting the mechanisms of Zn2þ dyshomeostasis in oligodendrocytes may offer novel therapeutic opportunities during excitotoxic damage to white matter.
MATERIALS AND METHODS Drugs Fura-2, FluoZin-3, 20 ,70 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), RhodZin-3, MitoTracker Green, nigericin, 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1), and 5-(and-6)-chloromethyl-20 70 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) were supplied by Invitrogen (Carlsbad, CA). N, N, N0 , N0 -tetrakis(2-pyridalmethyl)ethylenediamine (TPEN), 1, 10-phenanthroline, zinc pyrithione (ZnPy), Ca-EDTA, FCCP, rote-
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none, antimycin A, lonidamine, phenylarsine oxide, TEMPOL, uric acid, L-NAME, cyclosporin A, bongkrekic acid and 2,20 -dithiodipyridine (DTDP) were obtained from Sigma-Aldrich (Madrid, Spain). FeTMPyP and 3-morpholinosydnonimine (SIN-1) were provided by Cayman Chemical (Michigan, USA). a-amino3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA), cyclotiazide, and CNQX were ordered from Ascent (Cambridge, UK). Trolox was supplied by Calbiochem. Other chemicals were of the highest commercial grade available.
Animals Sprague Dawley rats were housed in the animal facility of the University of the Basque Country and maintained in a temperature- (21 6 1 C) and humidity- (55 6 10%) controlled room with a 12-h light-dark cycle. Food and water were available ad libitum. Experiments were carried out in accordance with the guidelines of The European Communities Council Directive 86/609/EEC and were approved by the Animal Research Ethical Committee of our institution.
Cell Cultures Primary cultures of oligodendrocytes derived from the optic nerves of 12-day-old rats were prepared as described previously (SanchezGomez et al., 2003). Cells were seeded into 24-well plates bearing 14-mm-diameter coverslips coated with 10 lg/mL poly-D-lysine at a density of 10,000 cells per well and maintained at 37 C and 5% CO2 in a chemically defined medium (Barres et al., 1992). Primary astrocyte cultures were prepared according to previously described procedures (McCarthy and de Vellis, 1980). Cortical neurons were isolated from E18 rat embryos as previously described (Ruiz et al., 2009).
Imaging of Intracellular Zn2þ, Ca2þ, and pH For intracellular [Zn2þ], [Ca2þ], and pH measurements, oligodendrocytes (1 DIV) were loaded for 5–10 min at 37 C with cell-permeant (acetoxymethyl, AM) dye derivatives of FluoZin-3 (1 lM), Fura-2 (5 lM), or BCECF (200 nM) in culture medium. Cells were subsequently washed for at least 10 min at room temperature in HBSS buffer containing 137 mM NaCl, 20 mM HEPES, 10 mM glucose, 4 mM NaHCO3 and 2 mM CaCl2 (pH 7.4). Ca2þ- and/or Naþ-free solutions were prepared by omitting CaCl2 and/or substituting Naþ with NMDG. Imaging of mitochondrial Zn2þ was carried out using the indicator RhodZin-3 AM, which is positively charged and follows the electrical gradient to accumulate in mitochondria, where it is retained on de-esterification (Sensi et al., 2003). Oligodendrocytes (1 DIV) were loaded with 0.5 lM RhodZin-3 AM for 15 min at 37 C and washed twice with culture medium. The residual cytosolic fraction of the dye was eliminated when the cells were kept in culture for an additional 18 h after loading, whereas the mitochondrial dye fluorescence was maintained. Experiments were performed in a coverslip chamber mounted on the stage of a Zeiss (Oberkochen, Germany) inverted epifluorescence microscope (Axiovert 35), equipped with a 150 W xenon lamp (Polychrome IV; T.I.L.L. Photonics, Martinsried, Germany) and a Plan Neofluar 40X oil immersion objective (Zeiss). Fura-2
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was excited alternately at 340/380 nm and FluoZin-3 at 490 nm. Emission of both dyes was monitored at 520 nm. Fluorescence images of RhodZin-3 were acquired using 550 nm excitation and 585 nm emission. [Ca2þ]i measurements were expressed as the ratio of F340/ F380, whereas FluoZin-3 and RhodZin-3 fluorescence was normalized to starting values and expressed as F/F0. In an attempt to calibrate FluoZin-3 fluorescence measurements, in some experiments [Zn2þ]i was determined as Kd [(F-Fmin)/(Fmax-F)] by using a Kd of 15 nM (Gee et al., 2002; Kiedrowski 2011). Fmax was obtained at the end of each experiment by adding the Zn2þ ionophore Na-pyrithione (25 lM) in the presence of 100 lM Zn2þ, and Fmin was obtained by adding the cell-permeable Zn2þ chelator TPEN (10 lM). Results from Fura-2 and FluoZin-3 assays were calculated as the area under the curve of the response for each cell from the start of the experiment. BCECF was excited at 440/490 nm and emission was monitored at 535 nm. Fluorescence F490/F440 ratios were converted to intracellular pH (pHi) according to the formula pH¼pKa -Log [(RmaxR)/(R-Rmin)], with a value of 7.135 used for pKa (Guia and Bose 2004). In situ calibration was carried out by exposing the cells to high (pH 9, Rmax) and low (pH 4, Rmin) pH in the presence of nigericin (10 lM) and high levels of potassium (140 mM). Images were acquired every 5 s with a high-resolution digital black/white CCD camera (ORCA; Hamamatsu Photonics Iberica, Barcelona, Spain) using the AquaCosmos software program (Hamamatsu Photonics Iberica). Background fluorescence was subtracted at the beginning of each experiment. Data were expressed as the mean 6 standard error of the mean (SEM) of the number of cells tested in at least three independent experiments, and the percentages of inhibition for each condition were always calculated versus control experiments carried out in parallel.
Confocal Imaging of Mitochondrial Zn2þ Oligodendrocytes were loaded with RhodZin-3 AM as described above, and incubated overnight in culture medium for elimination of residual cytosolic dye. At 2 DIV, cells were loaded with the mitochondrial probe MitoTracker Green FM (250 nM) for 15 min at 37 C and washed in HBSS buffer. Confocal images were obtained using a laser-scanning confocal microscope (Olympus Fluoview FV500) equipped with a 60X objective.
Quantitative PCR (qPCR) of Brain MTs Total RNA from cell cultures and rat tissue samples was extracted using TRIzol reagent according to the manufacturer’s recommendations (Invitrogen). First strand cDNA synthesis was carried out with reverse transcriptase SuperscriptTM III (Invitrogen) using random primers as previously described (Mato et al., 2009). Specific primers for MT-I, -II, and -III were designed with the PrimerExpress software (Applied Biosystems, Madrid, Spain) and were targeted to exon junctions to avoid amplification of contaminating genomic DNA. Primer sequences were as follows: rat MT-I forward, 5’-GAA GAG CTG CTG CTC CTG CTG-3’, and reverse, 5’-TGA GTT GGT CCG GAA ATT ATT TACA-3’; rat MT-II forward, 5’-AAC TCT GCA GCG ATC TCT CGT T-3’, and reverse, 5’-GAT CCA TCT GTG GCA CAG GAG-3’; rat MT-III forward, 5’-GGAACTAAGCTACAGTCTCTCGCG-3’, and reverse, 5’-GCA GGA ACC ACC
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AGT AGG ACA-3’; rat glyceraldehyde-3-phosphate dehydrogenase forward, 5’-GAA GGT CGG TGT CAA CGG ATT T-3’, and reverse, 5’-CAA TGT CCA CTT TGT CAC AAG AGA A-3’. Realtime qPCR was performed in an ABI Prism 7000 Sequence Detection System Instrument (Applied Biosystems) as previously described (Mato et al., 2009). For visualization of amplified DNA, PCR products were loaded on a 4% agarose gel containing SYBR Safe DNA stain (Invitrogen), which was electrophoresed for 90 min and visualized under UV light. Amplification resulted in single bands at 150, 84, and 81 bp corresponding to regions 159-308, 14-97 and 28-108 of the published sequences (NCBI accession numbers NM_138826.4, NM_001137564.1, and NM_053968.2) of rat MTI, MT-II and MT-III, respectively. For the comparison of MT expression levels between cell types, each experimental sample was assayed using three replicates for each primer, and the amount of cDNA was calculated from a standard curve of stock cDNA obtained from rat brain. The expression level of each gene was normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase as endogenous reference. The relative abundance of the MTs in oligodendrocyte cultures was determined by using the DCt method (see User Bulletin 2; Applied Biosystems). We verified that generated fluorescence was not overestimated by contamination from residual genomic DNA amplification (using RT negative controls), from primer dimer formation, or from external DNA contamination (via no template controls). qPCR products were also subjected to a dissociation protocol to ensure that a single amplicon of the expected melting temperature was indeed obtained.
Analysis of MT Expression in Optic Nerve Oligodendrocytes Double immunostaining of MT and oligodendrocyte markers was carried out in cell cultures and in optic nerve sections using specific antibodies against rat MT-I/II and MT-III, generously provided by Dr. J. Hidalgo (Carrasco et al., 1999). Live oligodendrocytes were incubated in vitro with a mouse monoclonal antibody against O4 (1:10, hybridoma supernatant), generously provided by Dr. C. Thomson, as previously described (Mato et al., 2009). Antibody R -conjugated anti-mouse labeling was visualized using Texas RedV IgM in phosphate-buffered saline containing 5% NGS. Cells were permeabilized with 0.1% Triton X-100þ5% NGS in phosphate-buffered saline for 30 min at room temperature, and subsequently incubated overnight at 4 C with rabbit antibodies against MT-I/II and MT-III diluted in blocking buffer. In tests of the ability of Zn2þ to induce MT-I/II and MT-III expression in oligodendrocytes, cells were incubated with 50-100 lM ZnSO4 for 24 h and fixed at 2 DIV. For the in situ analysis of MT localization, adult rats were deeply anesthetized with chloral hydrate (500 mg/kg, i.p.) and transcardially perfused with 0.1 M phosphate buffer (pH 7.4) followed by 4% paraformaldehyde in the same buffer. After extraction, optic nerves were cryoprotected in 15% sucrose and frozen. Cryostat sections (10 lm) were incubated in blocking solution containing 0.1% Triton X-100 plus 5% NGS in phosphate-buffered saline for 30 min at room temperature, and incubated with mouse anti-adenomatous polyposis coli (APC) antibody (2.5 lg/ml, Calbiochem, Darmstadt, Germany) diluted in blocking buffer for 1 h at 37 C. Sections were
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then incubated with goat anti-mouse Alexa 488 IgG (Molecular Probes) for 1 h at room temperature, and subsequently incubated with anti-MT-I/II and anti-MT-III antibodies overnight at 4 C. Following extensive washing, anti-MT antibodies were detected by incubation with goat anti-rabbit Alexa 488 IgG (Molecular Probes) for 1 h at room temperature. Hoechst 33258 (5 lg/ml; Sigma) was used for chromatin staining. Non-specific interactions of secondary antibodies were verified by omitting the primary antibodies. Coverslips and optic nerve sections were mounted in Prolong Gold Antifade Reagent (Invitrogen) and visualized using a laser-scanning confocal microscope (Olympus Fluoview FV500).
Cell Viability Assays Oligodendrocytes (1-2 DIV) were exposed to AMPA in the presence of 100 lM cyclotiazide for 30 min, and cell viability was determined 24 h after drug application. Oligodendroglial viability was assessed with 1 lM calcein-AM (Invitrogen) as described previously (Sanchez-Gomez et al., 2011). Calcein fluorescence was estimated using a Synergy-HT fluorimeter (Bio-Tek Instruments Inc., Beverly, MA). Results were expressed as percentage of cell death versus control, and at least three independent experiments in triplicate were performed for each condition.
Measurements of ROS Intracellular generation of ROS was determined via CM-H2DCFDA. Oligodendrocytes were exposed to AMPA as described, incubated in fresh medium for 30 min, and subsequently loaded with 30 lM CM-H2DCFDA for 30 min. Calcein-AM (1 lM) was used to quantify the number of cells within the reading field. Fluorescence was measured with a Synergy-HT fluorimeter. The excitation and emission wavelengths for CM-H2DCFDA and calcein were as suggested by the supplier. Results were expressed as the mean 6 SEM of at least three independent experiments carried out in triplicate.
Analysis of Mitochondrial Membrane Potential Oligodendrocyte cultures were exposed to AMPA alone or in the presence of TPEN for 30 min, and the changes in mitochondrial membrane potential were monitored by reduction of JC-1 according to the manufacturer’s protocol. Briefly, after drug treatment and washout (30 min), cells were loaded with 3 lM JC-1 in culture medium for 15 min at 37 C and were then washed with HBSS two times to eliminate excess dye. The monomeric form of JC-1 in the cytosol fluoresces green (emission read at 527 nm when excited at 485 nm), whereas highly concentrated JC-1 aggregates within the mitochondrial matrix fluoresce red (emission at 590 nm when excited at 485 nm). Both JC-1 monomers and aggregates were detected using a Synergy-HT fluorimeter, and the changes in mitochondrial potential were calculated as the red/green ratio for each condition. All experiments were performed at least in triplicate and plotted as mean 6 SEM.
Excitotoxicity in Isolated Optic Nerves Optic nerves of P12 rats were dissected in cold oxygen-saturated artificial CSF containing 126 mM NaCl, 3 mM KCl, 2 mM MgSO4, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2 mM CaCl2, and 10 mM glucose, as previously described (Sanchez-Gomez et al., 2003). Sub-
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sequently, the nerves were placed in a 48-well plate containing artificial CSF and incubated with 10 lM AMPA in the presence of 100 lM cyclotiazide for 1 h at 37 C and 5% CO2 in the presence of 10 lM TPEN, 25 lM CNQX, or vehicle. Following incubation, the media were replaced with fresh oxygen-saturated artificial CSF and incubated for another 2 h at 37 C. Tissue damage was analyzed by measuring lactate dehydrogenase (LDH) release in the incubation R medium 60 and 120 min post-stimulus, using the CytoTox 96V assay (Promega Biotech Iberica, Spain). At the end of the experiment, the optic nerves were homogenized by mechanical scraping in TM ice-cold RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Spain), and subjected to centrifugation (8000 rpm at 4 C for 10 min) to remove insoluble material. Experiments were performed in duplicate, normalized as a function of protein content, and expressed as the percentage of LDH release by control nerves within each assay.
Data Analysis In cytosolic Ca2þ, Zn2þ, and pH measurement experiments, n corresponds to the number of cells used for each condition; in the rest of the assays using oligodendrocytes in vitro, n corresponds to the number of cultures tested. In excitotoxicity assays with isolated optic nerves, n represents the number of animals tested. Data were analyzed with Excel (Microsoft, Seattle, WA) and Prism (GraphPad Software, San Diego, CA) software. Statistical significance between two datasets was tested using Student’s t-test, with a significance threshold of P < 0.05.
RESULTS AMPA Receptor Activation Triggers Ca2þDependent Elevation of Cytosolic Zn2þ Levels in Oligodendrocytes Activation of AMPA receptors with 10 lM AMPA in the presence of 100 lM cyclotiazide evoked rapid-onset elevation of FluoZin-3 fluorescence in rat optic nerve oligodendrocytes in vitro, an effect that was prevented by treatment with 25 lM CNQX (Fig. 1A,C). Consistent with an increase in intracellular Zn2þ, bath application of the membrane-permeable Zn2þ chelator TPEN (10 lM) at the end of AMPA exposure decreased FluoZin-3 fluorescence below basal levels (Fig. 1B). This effect was mimicked by another membrane-permeable Zn2þ chelator, namely 1,10-phenanthroline (100 lM, data not show). In situ calibration of several experiments as described in Materials and Methods suggested that intracellular Zn2þ elevations in oligodendroglial somata after 10 min of AMPA exposure may reach the high picomolar range (mean peak ¼ 695 6 0.098 pM; n ¼ 98 cells; Fig. 1B). Chelation of extracellular Zn2þ with 20 lM Ca-EDTA only marginally attenuated AMPA-induced FluoZin-3 fluorescence increases (8.4 6 3.9% inhibition; n ¼ 80 cells; p ¼ 0.14; Fig. 1C). By contrast, Ca-EDTA completely prevented the increase in FluoZin-3 fluorescence triggered by bath application of the Zn2þ ionophore ZnPy (Supporting Information Volume 000, No. 000
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FIGURE 1: Activation of AMPA receptors triggers Ca21-dependent mobilization of intracellular Zn21 in oligodendrocytes. (A, B) Representative images and time course depicting FluoZin-3 fluorescence increases evoked by exposure of oligodendrocytes to 10 lM AMPA plus 100 lM cyclotiazide for 10 min. Scale bar, 10 lm. Bath application of 10 lM TPEN at the end of the experiment decreased the FluoZin-3 signal below basal levels. The curve illustrates the mean 6 SEM responses of 98 cells. (C) Cytosolic Zn21 increases elicited by stimulation of AMPA receptors were not prevented by the extracellular Zn21 chelator Ca-EDTA (20 lM; n 5 80 cells), were potentiated by removal of extracellular Na1 (n 5 104 cells; ***P < 0.0001), and were strongly reduced in Ca21-free (n 5 85 cells; ***P < 0.0001) or Na1/Ca21-free buffers (n 5 63 cells; ***P < 0.0001). (D) Time courses showing FluoZin-3 (black) and Fura-2 (gray) responses evoked by exposure of oligodendrocytes to AMPA (10 lM plus 100 lM cyclotiazide applied for 10 min at the time indicated by the arrow), both in the presence (solid lines) and the absence (dashed lines) of extracellular Ca21. Fura-2 experiments were normalized and expressed as percent of baseline minus 100 for each time point. (E) Correlation between FluoZin-3 and Fura-2 responses elicited by bath application of AMPA (10 lM plus 100 lM cyclotiazide) under control or Ca21-, Na1-, or Ca21/Na1-free conditions (n 5 38–84 cells).
Fig. 1), showing that Ca-EDTA effectively chelates extracellular Zn2þ in our experimental conditions. Collectively, these data indicate that AMPA receptor activation triggers a process of fast intracellular Zn2þ mobilization in oligodendrocytes. Ca2þ influx is an important mechanism for the release of cytosolic Zn2þ pools in neurons (Dineley et al., 2008; Kiedrowski 2011; Kiedrowski 2012; Sensi et al., 2002). When analyzing the temporal course of AMPA-induced Ca2þ and Zn2þ responses in oligodendrocytes loaded with Fura-2 or FluoZin-3, we observed that the onset of sharp Fura-2 fluorescence rises occurred before the more progressive increases in FluoZin-3 signals (Fig. 1D), suggesting that cytosolic Ca2þ accumulation precedes Zn2þ deregulation upon AMPA exposure. In order to further analyze the Ca2þ-dependence of Zn2þ elevations in oligodendrocytes, we studied the relationship between Fura-2 and FluoZin-3 responses under various experimental conditions that are expected to modulate AMPA-induced Ca2þ signals. Removal of extracellular Ca2þ almost completely abolished the increase in cytosolic Ca2þ and Zn2þ evoked by activation of AMPA receptors (Fig. 1C,D), indicating that Ca2þ influx is responsible for AMPAinduced cytosolic Zn2þ mobilization. Conversely, AMPAMonth 2013
induced Ca2þ and Zn2þ responses were strongly potentiated in the absence of extracellular Naþ (Naþ substituted with NMDG); this effect was reversed when Ca2þ was omitted (Fig. 1C). As shown in Fig. 1E, we observed a strong correlation between AMPA-elicited intracellular Zn2þ and Ca2þ responses recorded in the same cultures (r2 ¼ 0.939; p ¼ 0.03). In contrast to the effect of TPEN (10 lM) on FluoZin-3 responses, bath application of the Zn2þ chelator under basal conditions or at the end of AMPA exposure did not substantially abolish Fura-2 signals (Supporting Information Fig. 2), indicating that changes in the fluorescence of the Ca2þ indicator were not due to Zn2þ. Taken together, these results suggest that fluctuation of cytosolic Ca2þ is the main mechanism underlying intracellular Zn2þ mobilization following AMPA receptor activation in oligodendrocytes. Mitochondrial and Protein-Bound Pools Contribute to AMPA Receptor-Triggered Intracellular Zn2þ Mobilization As previously reported in neurons (Sensi et al., 2003), the mitochondrial Zn2þ indicator RhodZin-3 AM exhibited a high degree of colocalization with the mitochondrial marker 5
FIGURE 2: Mitochondria contribute to AMPA-induced cytosolic Zn21 increases. (A) Oligodendrocytes were loaded with the mitochondrial Zn21 probe RhodZin-3 (red) at 1 DIV and with the mitochondrial marker MitoTracker Green (green) at 2 DIV. Confocal microscopy reveals a high degree of colocalization between the two fluorescent probes, suggesting the existence of mitochondrial Zn21 pools in oligodendrocyte cultures. Scale bar, 10 lm. (A0 ) Bath application of the membrane permeable Zn21 chelators TPEN (50 lM, n 5 18) and 1,10-phenanthroline (phen, 100 lM, n 5 16), and exposure to AMPA (10 lM plus 100 lM cyclotiazide, n 5 17), attenuated RhodZin-3 fluorescence below control levels (n 5 26). (B) Time course of the increase in FluoZin-3 fluorescence triggered by bath application of the mitochondrial protonophore FCCP (3 lM applied for 10 min at the time indicated by the arrow; n 5 62 cells) and chelation by 10 lM TPEN at the end of the experiment. (C, D) Time course and bar graph showing the inhibition of AMPA-induced FluoZin-3 responses (10 lM plus 100 lM cyclotiazide, applied for 10 min at the time indicated by the arrow) in oligodendrocytes preincubated with 3 lM FCCP (n 5 84 cells; **P < 0.01) and with the mPTP inhibitors cyclosporin A (CsA, 5 lM; n 5 101 cells; **P < 0.01) and bongkrekic acid (BA, 10 lM; n 5 61 cells; ***P < 0.0001).
Mitotracker Green in oligodendrocytes (Fig. 2A). Bath application of the membrane permeable Zn2þ chelators TPEN (50 lM) and phenanthroline (100 lM) attenuated RhodZin-3 fluorescence, confirming the existence of mitochondrial Zn2þ pools (2A0 ). Furthermore, exposure to AMPA also elicited a small decrease in RhodZin-3 fluorescence (2A0 ), indicating that mitochondrial Zn2þ pools contribute to cytosolic Zn2þ rises upon activation of AMPA receptors. To gain an insight into the mechanisms involved in AMPA-induced mobilization of mitochondrial Zn2þ, we studied the ability of different mitochondrial toxins to modulate cytosolic Zn2þ levels. Bath application of 3 lM FCCP led to a rapid rise in FluoZin-3 6
fluorescence that was sensitive to TPEN (Fig. 2B), suggesting that mitochondrial Zn2þ can be mobilized to the cytosol upon depolarization of the mitochondrial membrane. This effect was mimicked by the mitochondrial respitatory chain inhibitors rotenone (complex I, 20 lM) and antymicin A (complex III, 20 lM), the mitochondrial permeability transition pore (mPTP) inducer lonidamine (100 lM), and the SH group reagent phenylarsine oxide (5 lM), which induces mitochondria membrane depolarization and opening of the mPTP (data not shown). Preincubation of oligodendrocytes with FCCP or rotenone partially occluded subsequent AMPA-induced FluoZin-3 responses (Fig. 2C,D), providing Volume 000, No. 000
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further support for a mitochondrial contribution to cytosolic Zn2þ increases during AMPA receptor activation. We then addressed the possibility that opening of the mitochondrial permeability transition pore (mPTP) participates in AMPAtriggered Zn2þ mobilization. Consistent with such a mechanism, preincubation with the mPTP blockers cyclosporin A (5 lM) and bongkrekic acid (10 lM) attenuated AMPAinduced Zn2þ rises (Fig. 2C,D). Collectively, these data suggest that mitochondrial pools contribute to cytosolic Zn2þ increases during the activation of AMPA receptors in oligodendrocytes via mechanisms that involve depolarization of the mitochondrial membrane and opening of the mPTP. Another potential source of cytosolic Zn2þ during AMPA receptor activation is the protein-bound pool. In order to address this possibility, we first analyzed the expression of brain MT isoforms in optic nerve oligodendrocytes in vitro. Our data indicate that oligodendrocytes express MT-I, MT-II, and MT-III mRNA under basal culture conditions (Fig. 3A,B). Analysis of qPCR data suggested that the levels of MT-I mRNA in cultured oligodendrocytes were higher than the mRNA levels of MT-II and MT-III (Fig. 3B). Immunocytochemical analysis revealed that MT-I/II and MT-III are expressed in the somata and processes of oligodendrocytes in vitro (Fig. 3C), and we detected partial colocalization between MT-I/II and MT-III immunolabeling and APC-positive oligodendrocytes in the rat optic nerve (arrows in Fig. 3D). As reported in other cell types (West et al., 2008), expression of MT-I/II, but not MT-III, was strongly induced in oligodendrocyes in vitro following treatment with extracellular Zn2þ (data not shown). We next studied the effects of the disulfide-oxidizing agent DTDP, which induces Zn2þ release from MTs (Aizenman et al., 2000; Malaiyandi et al., 2004; Maret and Vallee 1998) in cultured oligodendrocytes. Bath application of DTDP (10 and 50 lM) elicited a concentration-dependent rise in FluoZin-3 fluorescence that was completely abolished by TPEN (Fig. 3E), consistent with a DTDP-mediated increase in cytosolic free Zn2þ levels. Finally, oligodendrocyte preincubation with DTDP reduced the size of AMPAevoked Zn2þ responses (inhibition was 16.7 6 5.3% and 57 6 2.4% following treatment with 10 and 50 lM DTDP, respectively; n ¼ 58-62 cells; P < 0.01 and P < 0.0001, respectively; Fig. 3F). These data suggest that release from MTs may contribute to the increase in cytosolic Zn2þ triggered by stimulation of AMPA receptors in oligodendrocytes. Glutamate-induced [Zn2þ]i rises in neurons are caused in part by Ca2þ-induced accumulation of ROS (Dineley et al., 2008). We next examined the possible contribution of oxidative stress to AMPA-induced [Zn2þ]i mobilization. Consistent with previous reports (Li et al., 2011a; Zhang et al., 2007), bath application of the peroxynitrite generator SIN-1 (500 lM) led to an increase in FluoZin-3 fluorescence (Fig. Month 2013
4A), further suggesting that optic nerve oligodendrocytes are sensitive to oxidant-induced intracellular Zn2þ release. The increase in [Zn2þ]i evoked by SIN-1 treatment was blocked by the peroxynitrite scavenger uric acid (1 mM), the decomposition catalyst of peroxynitrite FeTMPyP (10 lM), and the superoxide scavenger TEMPOL (1 mM; Fig. 4A,B). In contrast, none of these compounds counteracted AMPA-induced cytosolic Zn2þ mobilization in oligodendrocytes (Fig. 4B). Similarly, neither the antioxidant compound Trolox (1 mM) nor the nitric oxide synthase inhibitor L-NAME (1 mM), previously reported to reduce glutamate receptor-triggered toxicity in oligodendrocytes (Alberdi et al., 2006; MartinezPalma et al., 2003), inhibited intracellular Zn2þ rises following AMPA exposure (data not shown). Taken together, these observations indicate that ROS generation is not the primary mechanism underlying fast intracellular Zn2þ rises during stimulation of AMPA receptors in oligodendrocytes. Role of Cytosolic Acidification in AMPA Receptor-Induced Intracellular Zn2þ Rises Acidosis is a potent trigger of Zn2þ mobilization from MTs (Jiang et al., 2000; Sensi et al., 2003), and recent studies suggest that a pH drop promotes glutamate-induced Zn2þ increases in neurons (Kiedrowski, 2011,2012). Thus, we carried out a series of experiments to investigate the role of pH in Zn2þ homeostasis in oligodendrocytes. Modification of extracellular pH (pHe) from 7.4 to pH 6.4 and 8.4 induced rapid equilibration with the cytosol (pHi), measured as changes in the fluorescence ratio of the pH-sensitive probe BDECF (Fig. 5A). In addition, extracellular acidification triggered a fast-onset increase in FluoZin-3 fluorescence, whereas induction of alkaline pHi by exposure to a buffer at pH 8.4 led to a progressive decrease in basal FluoZin-3 fluorescence in oligodendrocytes (Fig. 5B). These observations are unlikely to result from direct pH effects on FluoZin-3, as the fluorescence response of this probe is not sensitive to changes in pH over a range from 6.0 to 9.0 (Gee et al., 2002). Regarding the role of pHi during AMPA receptor-triggered Zn2þ rises, exposure of oligodendrocytes to AMPA at pH 6.4 resulted in marked potentiation of FluoZin-3 responses, whereas application of a buffer at pH 8.4 elicited the opposite effect (Fig. 5C,D). These results are unlikely to reflect a direct effect of pHe on AMPA receptor functionality, as extracellular protons inhibit AMPA receptor-mediated responses (Ihle and Patneau 2000; Lei et al., 2001). However, previous evidence also indicates that extracellular acidosis potentiates Zn2þ entry through voltage-gated Ca2þ channels and AMPA receptors (Kerchner et al., 2000; Sensi et al., 2006). Ruling out the possibility that enhanced cytosolic Zn2þ increases upon AMPA exposure at acidic pHe may arise from extracellular sources, Ca-EDTA did not prevent 7
FIGURE 3: MT-bound pools contribute to AMPA-induced Zn21 rises. (A) Detection of mRNA encoding MT-I, MT-II and MT-III in cultured astrocytes (Ast), neurons (Neu), and oligodendrocytes (OL) by qPCR (n 5 3-4). (B) Relative expression of MT-I, MT-II and MT-III mRNA in oligodendrocyte cultures (n 5 4). (C) Labeling of MT-I/II and MT-III in O4-positive oligodendrocytes (1 DIV). Scale bar, 10 lm. (D) Double immunofluorescence for MT-I/II or MT-III (green) and the oligodendrocyte marker APC (red) in rat optic nerves. Arrows indicate positive expression of MTs in oligodendrocytes. Scale bar, 10 lm. (E) Time course depicting the increase in FluoZin-3 fluorescence elicited by exposure to DTDP (10 and 50 lM, applied for 5 min at the time indicated by the arrow; n 5 63 cells) and chelation with 10 lM TPEN at the end of the experiment. (F) Concentration-dependent inhibition of AMPA-triggered FluoZin-3 responses in oligodendrocytes preincubated with DTDP (10 and 50 lM; n 5 63 and 55 cells, respectively).
FluoZin-3 responses under these experimental conditions (Fig. 5D). Collectively, these results point to the activation of pH-dependent mechanisms of cytosolic Zn2þ release and/or clearance in oligodendrocytes during AMPA exposure. We then analyzed whether Ca2þ-dependent cytosolic acidification might serve as a trigger for Zn2þ deregulation 8
during AMPA exposure. In support of this idea, bath application of AMPA elicited a rapid decrease in BCECF fluorescence indicative of a drop in pHi (Fig. 5E). Reminiscent of the observations regarding the fluctuation of Ca2þ and Zn2þ levels during AMPA receptor activation, AMPA-induced cytosolic acidification was prevented by removal of extracellular Volume 000, No. 000
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FIGURE 4: AMPA-induced mobilization of intracellular Zn21 is independent of ROS production. (A, B) Bath application of the peroxynitrite generator SIN-1 (500 lM, for 4 min at the time indicated by the arrow) elicited an increase in FluoZin-3 fluorescence in oligodendrocytes. This effect was blocked by preincubation with the peroxynitrite scavenger uric acid (UA, 1 mM), the decomposition catalyst of peroxynitrite FeTMPyP (10 lM), and the superoxide scavenger TEMPOL (1 mM). Traces and bars represent mean 6 SEM responses of 37–63 cells; ***P < 0.0001. In contrast, preincubation with 1 mM uric acid, 10 lM FeTMPyP, or 1 mM TEMPOL did not inhibit AMPA-triggered FluoZin-3 fluorescence rises. (B) Bars represent mean 6 SEM responses of 58-85 cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Ca2þ, and was strongly potentiated in a Ca2þ-dependent manner when extracellular Naþ was eliminated (Fig. 5F). These observations suggest that intracellular acidosis promotes Zn2þ dyshomeostasis during activation of AMPA receptors in oligodendrocytes. Cytosolic Zn2þ Transients Contribute to Oligodendroglial Excitotoxicity Intracellular Zn2þ overload is an important mediator of neuronal and glial injury in a variety of pathological conditions (Sensi et al., 2009; Zhang et al., 2007). In the next set of experiments we sought to elucidate the possible contribution of Zn2þ deregulation to oligodendroglial excitotoxicity mediMonth 2013
ated by AMPA receptors. Exposure to AMPA (10 lM plus cyclotiazide 100 lM) for 30 min induced a decrease in cell viability that was significantly blocked by coincubation with 10 lM TPEN (29% inhibition), suggesting that cytosolic Zn2þ rises participate in the excitotoxicity initiated by submaximal activation of AMPA receptors (Fig. 6A). This protective effect was not observed following exposure to higher AMPA concentrations (100 lM in Fig. 6A), which trigger necrotic oligodendrocyte death (Sanchez-Gomez et al., 2003). Acute application of TPEN had no effect on cell viability in the absence of stimulus, but induced massive oligodendrocyte death following overnight incubation (data not shown). Previous reports from our laboratory indicate that optic nerve oligodendrocytes in situ are sensitive to AMPA receptormediated toxicity (Domercq et al., 2010; Sanchez-Gomez et al., 2003). To further confirm the role of Zn2þ in oligodendrocyte excitotoxicity, we investigated the effect of TPEN on glutamate receptor-mediated injury to intact optic nerves. Freshly isolated P12 rat optic nerves were exposed to AMPA (10 lM plus cyclotiazide 100 lM) in oxygen-saturated artificial CSF for 1 h, and cell damage was monitored by measuring the amount of LDH in the incubation media 60 and 120 min post-stimulus. Addition of AMPA induced a marked increase in LDH release (Fig. 6B) that was absent in optic nerves coincubated with 25 lM CNQX (LDH release was 90.3 6 14.4% of control; n ¼ 7), indicating that cell death was triggered by activation of AMPA receptors. Exposure to TPEN did not alter LDH release under control conditions, but it significantly reduced AMPAinduced cell death (Fig. 6B), suggesting that Zn2þ dyshomeostasis contributes to oligodendrocyte excitotoxicity in situ. Mild cytotoxic activation of AMPA receptors triggers apoptosis in oligodendrocytes via signaling mechanisms that involve an alteration of mitochondrial function and the generation of ROS (Sanchez-Gomez et al., 2003). To investigate whether cytosolic Zn2þ rises are causally related to these events, we determined both ROS production and mitochondrial depolarization induced by AMPA in the absence and presence of TPEN. Using the ROS indicator DCFDA, we observed that the increase in ROS levels 30 min after drug washout was completely abolished by addition of 10 lM TPEN during the cytotoxic stimulus (Fig. 6C). Similarly, depolarization of the mitochondrial membrane, which was monitored 30 min after AMPA washout using the fluorescent probe JC-1, was also significantly prevented in oligodendrocytes coincubated with TPEN (Fig. 6D). Exposure to TPEN had no effect on ROS generation or mitochondria membrane potential under basal conditions (data not shown). Cytosolic Ca2þ overload is a key mediator of mitochondrial depolarization and ROS generation upon cytotoxic activation of AMPA receptors in oligodendrocytes (SanchezGomez et al., 2003 and 2011). On the other hand, recent 9
FIGURE 5: Cytosolic acidification contributes to AMPA-induced Zn21 dyshomeostasis in oligodendrocytes. (A, B) Modulation of pHe induces rapid pHi equilibration and deregulation of basal cytosolic Zn21 levels in oligodendrocytes. Changes in pHi (A) were measured following exposure to bathing solution at pH 7.4, 6.4, and 8.4 for 2 min, using the pH-sensitive probe BDECF (n 5 42–56 cells; ***P < 0.0001). (B) Exposure to a buffer at acidic pHe (6.4, continuously applied at the time indicated by the arrow) triggered a fast-onset increase in FluoZin-3 fluorescence, whereas induction of alkaline pHi by bath application of a buffer at pH 8.4 evoked a progressive decrease of basal FluoZin-3 fluorescence in oligodendrocytes (n 5 63–84 cells). (C, D) Time course and bar graph showing the effects of pHe modulation on AMPA-triggered cytosolic Zn21 responses. Exposure to pHe 6.4 or 8.4 during AMPA application led to marked potentiation and inhibition of FluoZin-3 responses, respectively (n 5 39–81 cells; ***P < 0.0001). (E, F) Activation of AMPA receptors in oligodendrocytes triggers Ca21-dependent cytosolic acidification. Exposure to AMPA (10 lM plus 100 lM cyclotiazide, applied at the time indicated by the arrow) induced a rapid drop in pHi, measured as a decrease in BCECF fluorescence ratio. Figure F depicts the changes in cytosolic pH following 2 min of exposure to AMPA in a Ca21-containing medium, or in Ca21-free, Na1-free, or Na1/Ca21-free buffers (n 5 28–70 cells; ***P < 0.0001 versus control; ##P < 0.01 and ###P < 0.001 versus AMPA). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Mato et al.: Zinc Mediates AMPA Toxicity to Oligodendrocytes
FIGURE 6: Role of Zn21 in oligodendrocyte toxicity mediated by AMPA receptors. (A) Cytotoxicity elicited by low, but not high concentrations of AMPA (10 and 100 lM plus 100 lM cyclotiazide, 30 min) was significantly reduced in the presence of the Zn21 chelator TPEN (10 lM). *P < 0.05 compared with vehicle; n 5 3-6 cultures. (B) Excitotoxic damage to oligodendrocytes in the optic nerve, quantified by LDH release 60 and 120 min after exposure to AMPA (10 lM plus 100 lM cyclotiazide, 1 h), was reduced by coincubation with TPEN (**P < 0.01 and ***P < 0.0001 compared with vehicle; #P < 0.05 versus AMPA; n 5 9–11 animals per condition). (C, D) ROS generation and loss of mitochondrial membrane potential following mild stimulation of AMPA receptors were attenuated by TPEN. Oligodendrocyte production of ROS (C) and mitochondrial membrane potential (D) were determined by fluorimetry 30 min after AMPA exposure (10 lM plus 100 lM cyclotiazide, 30 min) using the dyes DCFDA (20 lM) and JC-1 (3 lM), respectively. *P < 0.05 compared with vehicle; n 5 3-4 cultures. (E) Time course of the effect of TPEN on AMPA-triggered intracellular Ca21 overload. Oligodendrocytes were exposed to AMPA (10 lM plus 100 lM cyclotiazide) for 9 min starting at min 1. Coapplication of TPEN did not alter the size of the cytosolic Ca21 rises evoked by sub-maximal activation of AMPA receptors (n 5 53 and 62 cells). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
evidence suggests that intracellular Zn2þ accumulation contributes to Ca2þ deregulation during oxygen-glucose deprivation in neurons (Medvedeva et al., 2009). We therefore examined the possibility that cytosolic Zn2þ increases participate in AMPA receptor-triggered Ca2þ responses in oligodendrocytes. In contrast to the above mentioned observations, the size of cytosolic Ca2þ increases following sub-maximal activation of AMPA receptors (10 lM plus cyclotiazide 100 lM) were not altered by coincubation with TPEN (Fig. 6E). Overall, these results suggest that cytosolic Zn2þ rises contribute to AMPA receptor-induced mitochondrial dysfunction, ROS generation, and oligodendrocyte cytotoxicity through mechanisms that do not involve the modulation of intracellular Ca2þ responses. Month 2013
DISCUSSION Ca2þ Dependency of AMPA-Induced Zn2þ Elevations in Oligodendrocytes One major aim of this study was to identify the primary mechanisms regulating intracellular Zn2þ concentration in oligodendrocytes exposed to AMPA. Our results indicate that cytosolic Ca2þ accumulation is the main trigger for Zn2þ rises upon activation of AMPA receptors. Supporting this conclusion, the time course of Fura-2 and FluoZin-3 fluorescence rises suggests that AMPA-induced cytosolic Ca2þ elevation precedes somatic Zn2þ increases. Although caution is needed when interpreting these results, as we did not simultaneously track changes in Ca2þ and Zn2þ in single oligodendrocytes, they are in line with previous observations regarding 11
the participation of Ca2þ in Zn2þ mobilization in neurons dually loaded with fluorescent indicators for both ions and exposed to glutamate (Kiedrowski 2011 and 2012). In addition, we observed that AMPA-induced FluoZin-3 responses were correlated with Fura-2 signals under a variety of experimental conditions expected to modulate cytosolic Ca2þ accumulation, such us the absence of extracellular Ca2þ, Naþ, or both. Since Fura-2 binds Zn2þ with high affinity (Grynkiewicz et al., 1985), a possible caveat to these results is that Zn2þ may actually account for an important component of the increases in the signal from the Ca2þ indicator. This possibility seems unlikely for several reasons. First, in agreement with previous estimations in neurons (Kiedrowski 2011), the present results suggest that intracellular concentrations of Zn2þ in oligodendrocytes, both under basal conditions and upon mild stimulation of AMPA receptors (present report), are 100- to 1000-fold lower than Ca2þ concentrations (Alberdi et al., 2002; Sanchez-Gomez et al., 2003). Second, Fura-2 signals were qualitatively unchanged by oligodendrocyte exposure to the Zn2þ chelator TPEN, which substantially abolished baseline and AMPA-induced FluoZin-3 fluorescence increases. Collectively, these data point to a negligible contribution of Zn2þ to Fura-2 responses under our experimental conditions, and support the idea that cytosolic Ca2þ fluctuations determine AMPA-induced Zn2þ deregulation in oligodendrocytes. Sources of AMPA Receptor-Triggered Zn2þ Rises in Oligodendrocytes Although the possibility that Zn2þ enters oligodendrocytes through AMPA and/or NMDA receptors in physiopathological conditions involving extracellular accumulation of the cation cannot be discarded (Sensi et al., 2011), the present investigation supports the hypothesis that at least two intracellular sources, the mitochondria and the protein-bound pool, contribute to Zn2þ dyshomeostasis following stimulation of AMPA receptors in these cells. In good agreement with previous observations in neurons (Dittmer et al., 2009; Sensi et al., 2003), we have demonstrated that Zn2þ accumulates in oligodendrocyte mitochondria under basal conditions, and can be released from these organelles upon depolarization of the mitochondrial membrane induced by the protonophore FCCP or by activation of glutamate receptors, as FCCP occluded subsequent Zn2þ rises following exposure to AMPA. In addition, we provide novel evidence that the mPTP participates in cytosolic Zn2þ overload during glutamate receptor activation, as incubation with the mPTP inhibitors cyclosporin A and bongkrekic acid counteracted AMPA-induced Zn2þ responses. Collectively, our results are consistent with a model in which Ca2þ overload upon AMPA receptor stimulation promotes cytosolic Zn2þ accumulation in oligodendrocytes 12
via mechanisms that involve the collapse of mitochondrial membrane potential and the opening of the mPTP. In addition to acting as a source of Zn2þ, converging lines of evidence suggest that mitochondria may also behave as a sink for deregulated Zn2þ levels in neurons (Dittmer et al., 2009; Medvedeva et al., 2009; Sensi et al., 2002). Thus, we cannot exclude the possibility that diminished Zn2þ uptake into mitochondria, likely resulting from membrane depolarization (Malaiyandi et al., 2005; Medvedeva et al., 2009), also contributes to Zn2þ deregulation during glutamate excitotoxicity to oligodendrocytes. Supporting the participation of protein-bound pools in glutamate receptor-induced Zn2þ dyshomeostasis, we have demonstrated that optic nerve oligodendrocytes express the MT-III isoform, as previously suggested in rat cortex (Miyazaki et al., 2002). In addition, our results provide evidence that these cells also express MT-I and MT-II mRNA and protein, both in vitro and in situ. Indeed, oligodendrocyte exposure to the disulfide-oxidizing agent DTDP led to a concentration-dependent elevation of cytosolic Zn2þ levels, indicative of release of the cation from MTs (Aizenman et al., 2000; Maret and Vallee 1998; Sensi et al., 2003). Finally, incubation with DTDP occluded subsequent Zn2þ increases by AMPA in a concentration-dependent manner, suggesting a common source of the released Zn2þ. Taken together, these data are consistent with the notion that oligodendrocytes possess a pool of Zn2þ bound to MTs, which is mobilized upon stimulation of AMPA receptors. It should be noted that despite the ability of MTs to release bound Zn2þ under physiopathological conditions involving oxidative stress and/or acidification, MTs are able to buffer excess cytosolic Zn2þ released from other intracellular compartments upon activation of glutamate receptors (Sensi et al., 2003). Further, there is good evidence that these proteins exert antioxidant and neuroprotective functions in a variety of brain disorders associated with excitotoxicity (West et al., 2008). Supporting the physiopathological relevance of MTs in oligodendrocytes, increased expression of MT-III has been reported in these cells following treatment with lipopolysaccharide (Miyazaki et al., 2002), as well as in multiple system atrophy (Pountney et al., 2011). In light of the present results, additional studies will be necessary to firmly establish the exact role of brain MT isoforms in oligodendroglial injury during excitotoxic damage to white matter. Cytosolic Acidification Mediates Zn2þ Dyshomeostasis in Oligodendrocytes Proposed mechanisms of intracellular Zn2þ mobilization in neurons include the generation of ROS and the acidification of the cytosol. Oxidation triggers Zn2þ release from MTs (Aizenman et al., 2000; Maret and Vallee 1998), and prior Volume 000, No. 000
Mato et al.: Zinc Mediates AMPA Toxicity to Oligodendrocytes
studies indicated that glutamate can mobilize Zn2þ through Ca2þ-dependent accumulation of ROS (Bossy-Wetzel et al., 2004; Dineley et al., 2008). In agreement with the hypothesis that oligodendrocytes are also susceptible to ROS-induced Zn2þ mobilization from intracellular pools, we reproduced previous observations regarding the ability of the peroxynitrite generator SIN-1 to promote cytosolic Zn2þ rises in these cells (Zhang et al., 2006; Li et al., 2011), an effect that was effectively prevented by various ROS scavengers. Nevertheless, none of the antioxidant compounds tested counteracted AMPA-induced cytosolic Zn2þ increases in oligodendrocytes, arguing against the possibility that fast generation of ROS is upstream of Zn2þ elevations within our experimental time window. In contrast, lowering and increasing pHi elicited dramatic potentiatory and inhibitory effects on AMPA-evoked Zn2þ rises, respectively. It is noteworthy that acidification of pHe elicited fast intracellular Zn2þ responses in the absence of additional stimuli, suggesting that cytosolic pH is a crucial mechanism of Zn2þ homeostasis in oligodendrocytes. This latest finding points to an important role of cytosolic Zn2þ deregulation as trigger of oligodendroglial damage in physiopathological situations that involve extracellular acidosis, such as hypoxia-ischemia. Finally, a causal relationship between cytosolic acidification and AMPA-induced Zn2þ mobilization in oligodendrocytes can be inferred from the observation that AMPA evoked a Ca2þ-dependent drop in pHi. Based on the present data and previous observations (Frazzini et al., 2007; Kiedrowski 2011 and 2012), the emerging scenario is that changes in cytosolic pH are the main trigger for fast intracellular Zn2þ mobilization upon stimulation of ionotropic glutamate receptors. The precise molecular mechanisms that determine acidosis-dependent Zn2þ deregulation during oligodendrocyte excitotoxicity may involve a destabilization of ion binding to MTs (Jiang et al., 2000), as well as an increased Zn2þ efflux from intracellular organelles bearing Hþ-dependent transporters such as ZnTs (Sekler et al., 2007), whose expression has not yet been characterized in these cells. Cytosolic Zn2þ Rises Are Early Triggers of Excitotoxic Injury to Oligodendrocytes Importantly, the accumulation of intracellular Zn2þ following activation of oligodendrocyte glutamate receptors contributes significantly to cell damage, as evidenced by the ability of TPEN to prevent AMPA excitotoxicity both in vitro and in situ. From the mechanistic point of view, our results provide a link between cytosolic Ca2þ overload and mitochondrial depolarization upon activation of AMPA receptors in oligodendrocytes, adding to the growing body of evidence that Zn2þ is a critical mediator of mitochondrial dysfunction in the progression to cell death (Sensi et al., 2009). As previously reported in neurons (Bossy-Wetzel et al., 2004; Dineley Month 2013
et al., 2005), we found that Zn2þ is an important trigger for the generation of ROS during glutamate excitotoxicity to oligodendrocytes. Whether this effect is a direct consequence of the alteration of mitochondrial function by free Zn2þ (Sensi et al., 2003), or is secondary to the activation of specific signaling pathways leading to cytosolic oxidative stress in oligodendrocytes, such as protein kinase C (Li et al., 2011a) or extracellular signal-regulated kinase 42/44 (Zhang et al., 2006), remains to be established. Conclusions/Future Directions In conclusion, this study shows for the first time that destabilization of Zn2þ homeostasis participates in excitotoxic injury to oligodendrocytes, providing new insights into the mechanisms by which excessive activation of glutamate receptors triggers white matter damage. Activation of AMPA receptors induces Zn2þ release from intracellular pools, which precedes and mediates key cellular events associated with oligodendrocyte cytotoxicity, including mitochondrial depolarization and ROS production. An important question that remains to be addressed is the extent to which cytosolic Zn2þ accumulation contributes to the activation of excitotoxic injury cascades in oligodendrocytes. Second, although the present observations point to an important role of cytosolic acidification as a trigger for Zn2þ rises in oligodendrocytes, the relative contributions of MTs and Hþ-dependent or -independent transporters to cytosolic Zn2þ accumulation and clearance following activation of glutamate receptors requires further analysis. Detailed characterization of the molecular mechanisms responsible for Zn2þ dyshomeostasis in oligodendrocytes will possibly provide new pharmacological targets for therapeutic intervention in excitotoxic white matter injury.
Acknowledgments Susana Mato and Ana Bernal-Chico are recipients of a Ramon y Cajal contract and a fellowship from the University of the Basque Country-UPV/EHU, respectively. The authors thank J. Hidalgo for providing anti-MT-I/II and MT-III antibodies, and for helpful comments. The technical assistance of Hazel Gomez, Saioa Marcos, and the staff of the animal facility of the University of the Basque Country is gratefully acknowledged.
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