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• W2061460906 abstract "Cyclophilin d (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca2+, and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca2+ caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca2+ in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca2+ deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli. Cyclophilin d (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca2+, and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca2+ caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca2+ in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca2+ deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli. In 2005, four independent groups reported the beneficial effect of cyclophilin D (cypD) 2The abbreviations used are: cypD, cyclophilin D; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; cys A, cyclosporin A; ΔΨm, mitochondrial membrane potential; PTP, permeability transition pore; TR, thinness ratio. absence in transgenic mice, for an array of in vitro and in vivo pathologic stimuli (1Baines C.P. Kaiser R.A. Purcell N.H. Blair N.S. Osinska H. Hambleton M.A. Brunskill E.W. Sayen M.R. Gottlieb R.A. Dorn G.W. Robbins J. Molkentin J.D. Nature. 2005; 434: 658-662Crossref PubMed Scopus (1866) Google Scholar, 2Nakagawa T. Shimizu S. Watanabe T. Yamaguchi O. Otsu K. Yamagata H. Inohara H. Kubo T. Tsujimoto Y. Nature. 2005; 434: 652-658Crossref PubMed Scopus (1372) Google Scholar, 3Schinzel A.C. Takeuchi O. Huang Z. Fisher J.K. Zhou Z. Rubens J. Hetz C. Danial N.N. Moskowitz M.A. Korsmeyer S.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 12005-12010Crossref PubMed Scopus (713) Google Scholar, 4Basso E. Fante L. Fowlkes J. Petronilli V. Forte M.A. Bernardi P. J. Biol. Chem. 2005; 280: 18558-18561Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar). Ever since, the contribution of cypD in a variety of diseases has been strongly suggested or proven (for review, see Ref. 5Giorgio V. Soriano M.E. Basso E. Bisetto E. Lippe G. Forte M.A. Bernardi P. Biochim. Biophys. Acta. 2010; 1797: 1113-1118Crossref PubMed Scopus (148) Google Scholar), a momentum that was assisted by the wide availability of cypD knock-out (KO) mice. These studies converged to the conclusion that cypD-mediated mitochondrial permeability transition pore (PTP) regulates some forms of necrotic, but not apoptotic death. The notion in which PTP is involved in necrosis but not apoptosis has been originally suggested by the group of Crompton and colleagues (6Li Y. Johnson N. Capano M. Edwards M. Crompton M. Biochem. J. 2004; 383: 101-109Crossref PubMed Scopus (136) Google Scholar). A major difference among prerequisites for the manifestation of necrosis versus apoptosis is energy availability; a sufficient decline in energy reserves, primarily in ATP concentration, is a switch for a cell to die by necrosis rather than apoptosis (7Leist M. Single B. Castoldi A.F. Kühnle S. Nicotera P. J. Exp. Med. 1997; 185: 1481-1486Crossref PubMed Scopus (1651) Google Scholar, 8Volbracht C. Leist M. Nicotera P. Mol. Med. 1999; 5: 477-489Crossref PubMed Google Scholar). Such an extensive decrease in ATP is invariably associated with loss of mitochondrial membrane potential, ΔΨm (9Chinopoulos C. Adam-Vizi V. Biochim. Biophys. Acta. 2010; 1802: 221-227Crossref PubMed Scopus (88) Google Scholar, 10Chinopoulos C. Gerencser A.A. Mandi M. Mathe K. Töröcsik B. Doczi J. Turiak L. Kiss G. Konràd C. Vajda S. Vereczki V. Oh R.J. Adam-Vizi V. FASEB J. 2010; 24: 2405-2416Crossref PubMed Scopus (75) Google Scholar). Mindful of the large increases in intracellular Ca2+ during cell injury (11Chinopoulos C. Adam-Vizi V. FEBS J. 2006; 273: 433-450Crossref PubMed Scopus (203) Google Scholar) and the loss of ΔΨm preceding cell death (12Fiskum G. Starkov A. Polster B.M. Chinopoulos C. Ann. N.Y. Acad. Sci. 2003; 991: 111-119Crossref PubMed Scopus (220) Google Scholar), the conundrum appears that excessive Ca2+ induces PTP under conditions unfavorable for electrophoretic Ca2+ uptake by mitochondria (13Vajda S. Mándi M. Konràd C. Kiss G. Ambrus A. Adam-Vizi V. Chinopoulos C. FEBS J. 2009; 276: 2713-2724Crossref PubMed Scopus (13) Google Scholar). Some studies address this by proposing that in ischemia-reperfusion, Ca2+-induced PTP occurs during reperfusion of the affected tissue, but in several experimental models mimicking pathology, mitochondrial damage caused by excessive Ca2+ uptake did not involve restoration of bioenergetic functions. Partial resolution of this apparent contradiction came from an insightful work by the group of Bernardi demonstrating that the threshold for PTP induction by Ca2+ is modulated by the proton electrochemical gradient (14Bernardi P. J. Biol. Chem. 1992; 267: 8834-8839Abstract Full Text PDF PubMed Google Scholar, 15Petronilli V. Cola C. Bernardi P. J. Biol. Chem. 1993; 268: 1011-1016Abstract Full Text PDF PubMed Google Scholar, 16Petronilli V. Cola C. Massari S. Colonna R. Bernardi P. J. Biol. Chem. 1993; 268: 21939-21945Abstract Full Text PDF PubMed Google Scholar, 17Petronilli V. Costantini P. Scorrano L. Colonna R. Passamonti S. Bernardi P. J. Biol. Chem. 1994; 269: 16638-16642Abstract Full Text PDF PubMed Google Scholar, 18Scorrano L. Petronilli V. Bernardi P. J. Biol. Chem. 1997; 272: 12295-12299Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Specifically, they have shown that the more depolarized mitochondria are, the higher the likelihood that they will exhibit PTP induced by Ca2+. Later on, the same group extended its findings by showing that pyridine nucleotides and dithiol oxidation of specific sites also modulate the pore (19Costantini P. Chernyak B.V. Petronilli V. Bernardi P. J. Biol. Chem. 1996; 271: 6746-6751Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) and that electron flow through complex I is a modulator of PTP opening upon Ca2+ uptake (20Fontaine E. Eriksson O. Ichas F. Bernardi P. J. Biol. Chem. 1998; 273: 12662-12668Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), concepts with inherent connection to the proton electrochemical gradient. Brain mitochondria in relation to Ca2+-induced PTP deserve further attention, primarily because they reside within excitable cells exhibiting ample routes to Ca2+ and because unlike liver or heart mitochondria, there is still no universally accepted consensus here: claims of Ca2+ inducing PTP in brain mitochondria range from a partial (21Kristián T. Weatherby T.M. Bates T.E. Fiskum G. J. Neurochem. 2002; 83: 1297-1308Crossref PubMed Scopus (64) Google Scholar) to a complete effect (22Hansson M.J. Månsson R. Mattiasson G. Ohlsson J. Karlsson J. Keep M.F. Elmér E. J. Neurochem. 2004; 89: 715-729Crossref PubMed Scopus (62) Google Scholar), and the disagreement extends to the degree of cyclosporin A (cys A) sensitivity (11Chinopoulos C. Adam-Vizi V. FEBS J. 2006; 273: 433-450Crossref PubMed Scopus (203) Google Scholar, 22Hansson M.J. Månsson R. Mattiasson G. Ohlsson J. Karlsson J. Keep M.F. Elmér E. J. Neurochem. 2004; 89: 715-729Crossref PubMed Scopus (62) Google Scholar, 23Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar, 24Chinopoulos C. Starkov A.A. Fiskum G. J. Biol. Chem. 2003; 278: 27382-27389Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Furthermore, because experimental conditions strongly shape the outcome and characteristics of brain mitochondrial PTP (11Chinopoulos C. Adam-Vizi V. FEBS J. 2006; 273: 433-450Crossref PubMed Scopus (203) Google Scholar, 25Chinopoulos C. Adam-Vizi V. FEBS J. 2010; 277: 3637-3651Crossref PubMed Scopus (54) Google Scholar), it becomes imperative to investigate PTP in mitochondria within neurons and astrocytes. In the present study we have identified bioenergetic conditions in isolated brain mitochondria that allow the demonstration of a cypD dependence upon Ca2+-induced PTP opening and applied them to in situ neuronal and astrocytic mitochondria. C57BL/6J WT and KO for cypD littermate mice were a gift from Drs. Nika Danial and Anna Schinzel, from Howard Hughes Medical Institute and Dana-Farber Cancer Institute, Harvard Medical School. Mice were cross-bred for eight generations prior to harvesting brain tissues from WT and KO age-matched animals for the purpose of mitochondrial isolation and culturing of neurons and astrocytes. Nonsynaptic brain mitochondria from adult male WT and KO for cypD mice (aged 87–115 days) were isolated on a Percoll gradient as described previously (26Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (344) Google Scholar) with minor modifications detailed in Ref. 24Chinopoulos C. Starkov A.A. Fiskum G. J. Biol. Chem. 2003; 278: 27382-27389Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar. All animal procedures were carried out according to the local animal care and use committee (Egyetemi Allatkiserleti Bizottsag) guidelines. Mitochondria-dependent removal of medium Ca2+ was followed using the impermeant hexapotassium salt of the fluorescent dye Calcium Green 5N (Molecular Probes, Portland, OR). Calcium Green 5N (500 nm) was added to a 2-ml medium containing mitochondria (0.125 mg/ml) and 120 mm KCl, 10 mm Tris, 5 mm KH2PO4, 1 mm MgCl2, pH 7.6. Substrates were added where indicated. All experiments were performed at 37 °C. Fluorescence intensity was measured in a Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) using 517-nm excitation and 535-nm emission wavelengths. Swelling of isolated mitochondria was assessed by measuring light scatter at 520 nm in a GBC UV/visible 920 spectrophotometer. Mitochondria were added at a final concentration of 0.125 mg/ml to 2 ml of medium containing 120 mm KCl, 10 mm Tris, 5 mm KH2PO4, 1 mm MgCl2, pH 7.6. Substrates were added where indicated. At the end of each experiment, the nonselective pore-forming peptide alamethicin (40 μg) was added as a calibration standard to cause maximal swelling. All experiments were performed at 37 °C. ΔΨm was estimated fluorometrically with safranine O (27Akerman K.E. Wikström M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). Mitochondria (0.25 mg) were added to 2 ml of incubation medium containing 120 mm KCl, 10 mm Tris, 5 mm KH2PO4, 1 mm MgCl2, pH 7.6, and 2.5 μm safranine O. Fluorescence was recorded in a Hitachi F-4500 spectrofluorometer at a 5-Hz acquisition rate, using 495- and 585-nm excitation and emission wavelengths, respectively. Experiments were performed at 37 °C. To convert safranine O fluorescence into millivolts, a voltage-fluorescence calibration curve was constructed. To this end, safranine O fluorescence was recorded in the presence of 2 nm valinomycin and stepwise increasing K+ (in the 0.2–120 mm range) which allowed calculation of ΔΨm by the Nernst equation assuming a matrix K+ = 120 mm (27Akerman K.E. Wikström M.K. FEBS Lett. 1976; 68: 191-197Crossref PubMed Scopus (671) Google Scholar). Visualization of isolated mitochondria under epifluorescence imaging (Nikon Plan Fluor 100 × 1.3 NA) was achieved by loading mitochondria with fura-2/AM (8 μm for 20 min at 30 °C). Mitochondria were diluted to 1 mg/ml, and 5 μl was dropped on a coverslip, allowed to stand for 4 min prior to starting the perfusion. Image sequences (10 s/ratio frame, 50-ms exposure time, 2 × 2 binning) were acquired using an Micromax cooled digital CCD camera (Princeton Instruments) mounted on a Nikon Diaphot 200 inverted microscope (Nikon Corp., Tokyo, Japan). Image acquisition was controlled by Metafluor 3.5 (Universal Imaging Corp., West Chester, PA). The perfusate (120 mm KCl, 10 mm Tris, 5 mm KH2PO4, 1 mm MgCl2, pH 7.6, 50 ml/h flow rate) was temperature-controlled at 37 °C. Mixed primary cultures of cortical neurons and astrocytes were prepared from cypD-KO or wild-type (WT) mice pups (P 0–1). Cells were grown on poly-l-ornithine-coated 8-well LabTek II chambered coverglasses (Nunc, Rochester, NY) for 7–12 days, at a density of ∼105 cells/well in Neurobasal medium containing 2% B27 supplement and 2 mm glutamine (Sigma). Cultures were transfected at 7–9 days with mito-DsRed2 using Lipofectamine 2000 (Invitrogen) in Neurobasal medium at a 3:2 ratio of Lipofectamine (μl) to plasmid DNA (μg). Experiments were carried out at day 1–2 after transfection. Typical transfection rates were low, and therefore individual, nonoverlapping cells were visualized. Time lapse epifluorescence microscopy was carried out to image cells expressing mito-DsRed2 at 37 °C without superfusion in a medium containing 120 mm NaCl, 3.5 mm KCl, 1.3 mm CaCl2, 20 mm HEPES, 15 mm glucose at pH 7.4. For some experiments (detailed below) neurons were loaded with fura-FF/AM (2 μm) for 20 min before imaging. Experiments were performed on an Olympus IX81 inverted microscope equipped with a 60 × 1.4 NA oil immersion lens, a Bioprecision-2 xy-stage (Ludl Electronic Products Ltd., Hawthorne, NY), and a 75W xenon arc lamp (Lambda LS; Sutter Instruments, Novato, CA). For DsRed2 an 535/20 nm exciter, a 555LP dichroic mirror, and an 570LP emitter (Omega Optical, Brattleboro, VT) were used. Time lapses of z-series of 16 planes of 512 × 512-pixel frames (digitized at 14bit with no binning, 250-ms exposure time, yielding 0.1-μm pixel size and 0.8-μm z-spacing) were acquired using an ORCA-ER2 cooled digital CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) under control of MetaMorph 6.0 software (Molecular Devices, Sunnyvale, CA). For fura-FF a 340/26-nm and a 387/11-nm exciter (Semrock, Rochester, NY), a 405LP dichroic mirror, and a 475LP emitter (Omega Optical) were used, and single plane images were recorded after each DsRed2 z-stack. Mitochondrial swelling was measured by the thinness ratio (TR) technique (28Gerencser A.A. Doczi J. Töröcsik B. Bossy-Wetzel E. Adam-Vizi V. Biophys. J. 2008; 95: 2583-2598Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, the TR technique measures changes of average diameters of thread-like or punctate structures in fluorescence images using a pair of (high and low frequency) bandpass spatial filters. A calibration image series of mito-DsRed2 fluorescence showing mitochondrial swelling by valinomycin (200 nm) was recorded and used to train a spatial bandpass filter set in Image Analyst MKII (Image Analyst Software, Novato, CA). To calculate TR, for each time point the z-stack was mean intensity-projected, and the projection image was duplicated. Then, both images were spatially filtered, and the absolute value of pixels was taken. The TR was calculated as the ratio of the average fluorescence intensity in the high frequency bandpass-filtered over the low frequency bandpass-filtered image. Mitochondrial swelling causes the loss of high spatial frequency image details, therefore a decrease in the TR value. Base-line normalized TR is given as δTR = (TR − TR0)/TR0. Isolated brain and liver mitochondria were solubilized at a concentration of 10 mg/ml in a buffer containing 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, plus a mixture of protease inhibitors (Protease Inhibitor Mixture Set I; Calbiochem). Solubilized mitochondria were rapidly frozen and stored at −70 °C until further manipulation. Upon thawing, the protein concentration of the samples was estimated by the Bradford assay. Subsequently, samples were mixed with 50 mm dithiothreitol and NuPage loading buffer (Invitrogen) and heated to 70° for 10 min. The samples were then loaded (25 μg/lane) on a 4–12% BisTris gel, and separated by SDS-PAGE in the presence of N,N-dimethylformamide and sodium bisulfite (10% w/v). Separated proteins were transferred to a methanol-activated polyvinylidene difluoride membrane. Immunoblotting was performed as recommended by the manufacturer. Mouse monoclonal anti-cypD (Mitosciences, Eugene, OR) primary antibody was used at 1 μg/ml. Immunoreactivity was detected using the appropriate peroxidase-linked secondary antibodies (1:10,000; Jackson Immunochemicals Europe, Cambridgeshire, UK) and enhanced chemiluminescence detection reagent (ECL system; Amersham Biosciences). Upon completion of Western blotting the blots were stained with Ponceau S (0.5% Ponceau S (w/v) in 5% acetic acid), and loading of individual lanes was assessed by densitometric analysis (Scion Image, version alpha 4.0.3.2; Scion Corp., Frederick, MD). Standard laboratory chemicals, cys A, calcimycin, and alamethicin were from Sigma. Ru360 was from Calbiochem. SF 6847 was from BIOMOL. Calcium Green 5N 6K+ salt was from Invitrogen. Mito-DsRed2 was purchased from Clontech. Mitochondrial substrate stock solutions were dissolved in bi-distilled water and titrated to pH 7.0 with KOH. Electrophoretic Ca2+ uptake for induction of PTP is allowed either in the presence of respiratory substrates or in a substrate-free medium containing KSCN; diffusion of the lipophilic SCN− anion provides the driving force for electrophoretic Ca2+ accumulation (29Selwyn M.J. Dawson A.P. Dunnett S.J. FEBS Lett. 1970; 10: 1-5Crossref PubMed Scopus (99) Google Scholar, 30Nicolli A. Petronilli V. Bernardi P. Biochemistry. 1993; 32: 4461-4465Crossref PubMed Scopus (152) Google Scholar). However, in the original studies by Hunter and Haworth it was shown that PTP can be induced by Ca2+ in the absence of respiratory substrates (31Hunter D.R. Haworth R.A. Arch. Biochem. Biophys. 1979; 195: 453-459Crossref PubMed Scopus (603) Google Scholar), a phenomenon that has been subsequently reproduced (32Yamamoto T. Yoshimura Y. Yamada A. Gouda S. Yamashita K. Yamazaki N. Kataoka M. Nagata T. Terada H. Shinohara Y. J. Bioenerg. Biomembr. 2008; 40: 619-623Crossref PubMed Scopus (8) Google Scholar, 33Rigobello M.P. Turcato F. Bindoli A. Arch. Biochem. Biophys. 1995; 319: 225-230Crossref PubMed Scopus (46) Google Scholar, 34Moore G.A. Jewell S.A. Bellomo G. Orrenius S. FEBS Lett. 1983; 153: 289-292Crossref PubMed Scopus (82) Google Scholar), reviewed in (35Leverve X.M. Fontaine E. IUBMB Life. 2001; 52: 221-229Crossref PubMed Scopus (39) Google Scholar). Furthermore, it was also shown that substrates delay Ca2+-induced PTP (33Rigobello M.P. Turcato F. Bindoli A. Arch. Biochem. Biophys. 1995; 319: 225-230Crossref PubMed Scopus (46) Google Scholar, 34Moore G.A. Jewell S.A. Bellomo G. Orrenius S. FEBS Lett. 1983; 153: 289-292Crossref PubMed Scopus (82) Google Scholar), in accordance to the Bernardi scheme mentioned above. To reproduce these findings for our studies, isolated brain mitochondria were challenged by CaCl2, in the presence and absence of glutamate and malate, and light scattering was recorded spectrophotometrically at 520 nm. A three-pulse CaCl2 protocol was used for this and all subsequent similar experiments: 20 μm CaCl2 was given at 100 s, followed by 200 μm CaCl2 at 300 s and again at 500 s. Orange lines appearing in Figs. 1, 2, and 4 are control trace lines obtained from WT mitochondria not exposed to CaCl2. As shown in Fig. 1A, addition of 20 μm CaCl2 to substrate-supplemented or substrate-starved brain mitochondria of WT mice did not cause a decrease in light scatter; instead, a cessation in the initial base-line decrease was observed. However, the subsequent 200 μm CaCl2 pulse induced a large decrease in light scatter in substrate-starved but not substrate-supplemented mitochondria. The next 200 μm CaCl2 pulse given at 500 s did not induce any further changes in substrate-starved mitochondria, but caused a decrease in substrate-supplemented mitochondria. As shown in Fig. 1B, the effect of the first 200 μm CaCl2 pulse was cys A-sensitive; however, the second addition of 200 μm CaCl2 overrode the protective effect of cys A, consistent with the findings by Brustovetsky and Dubinsky (23Brustovetsky N. Dubinsky J.M. J. Neurosci. 2000; 20: 8229-8237Crossref PubMed Google Scholar). In Fig. 1B the effect of the pore-forming peptide alamethicin is also shown, so that the extent of changes in light scatter induced by CaCl2 can be better appreciated compared with maximum changes. Subsequent experiments benefitted from the availability of cypD-KO mice. We isolated mitochondria from the brains of WT and cypD-KO mice (see Fig. 1C). As shown in Fig. 1D, results obtained from substrate-starved mitochondria from cypD-KO mice were strikingly similar to those obtained from cys A-treated WT mice (Fig. 1B). The presence of substrates, however, did not provide additional protection in the cypD-KO mitochondria (Fig. 1E). Maximum swelling rates pooled from all experiments (expressed as percentage of swelling rate/minute and accounting for the condition producing the highest swelling rate as “maximum”) for each condition and after each Ca2+ pulse is shown in Fig. 1G. These results are in accord with earlier reports on various types of mitochondria and conditions, showing that high Ca2+ loads can induce PTP in the absence of substrates. In our hands, absence of substrates prevented isolated mitochondria from building a membrane potential of higher than −10 mV (data not shown). At this ΔΨm value, mitochondrial Ca2+ uptake is unfavorable (13Vajda S. Mándi M. Konràd C. Kiss G. Ambrus A. Adam-Vizi V. Chinopoulos C. FEBS J. 2009; 276: 2713-2724Crossref PubMed Scopus (13) Google Scholar). Indeed, recordings of extramitochondrial Ca2+ by Calcium Green 5N revealed that in the absence of substrates (Fig. 1F, traces c, d, and e) mitochondria were unable to perform Ca2+ sequestration, yet exhibited large changes in light scatter. Electron microscopy imaging of mitochondria that exhibited large changes in light scatter confirmed that this was due to swelling (data not shown). We therefore considered the possibilities that Ca2+ was either inducing cypD-sensitive swelling by acting on an extramitochondrial site, or because high amounts of CaCl2 were required, Ca2+ was entering mitochondria simply by a chemical gradient.FIGURE 2Effect of SF 6847 and/or inhibitors of the Ca2+ uniporter on mitochondrial Ca2+ uptake; modulation by cyclosporin A or genetic deletion of cyclophilin D. Traces of light scatter recorded spectrophotometrically at 520 nm during CaCl2 additions at the concentrations are indicated in the panels, to mitochondrial suspensions. Conditions of the suspensions are given in the panels. All panels are aligned on the x axis. Results are representative of at least four independent experiments. E, maximum swelling rates pooled from all individual experiments (expressed as in Fig. 1) for each condition and after each Ca2+ pulse. Error bars represent S.E.; a is statistically significant from b, c, d, e, f, and g, p < 0.001; d is statistically significant from e, p < 0.05, one-way ANOVA on Ranks.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Effect of respiratory chain inhibition on Ca2+-induced PTP. Traces of light scatter recorded spectrophotometrically at 520 nm during CaCl2 additions at the concentrations indicated in the figures, to mitochondrial suspensions are shown. All experiments were performed on WT mice A: a, no substrates; b, plus glutamate plus malate; c, plus glutamate plus malate plus 1 μm rotenone; d, no substrates + 1 μm rotenone; e, no substrates + 1 mm KCN; f, plus glutamate plus malate plus 1 mm KCN. B: a, no substrates; b, no substrates + 1 μm piericidin A; c, no substrates + 1 μm rotenone. C: a, no substrates; b, no substrates + 0.2 μm stigmatellin; c, no substrates + 2 μm stigmatellin; d, no substrates + 0.5 μm myxothiazol; e, no substrates + 10 μm myxothiazol; f, no substrates + 1 mm KCN. Results are representative of at least four independent experiments. D, maximum swelling rates pooled from all individual experiments (expressed as in FIGURE 1, FIGURE 2) for each condition and after each Ca2+ pulse. Error bars represent S.E.; a is statistically significant from c, p < 0.05; b is statistically significant from d, p < 0.05, one-way ANOVA on Ranks.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To address the site of action of Ca2+ on the light scatter, we pretreated mitochondria with the Ca2+ uniporter inhibitor, Ru360 (36Matlib M.A. Zhou Z. Knight S. Ahmed S. Choi K.M. Krause-Bauer J. Phillips R. Altschuld R. Katsube Y. Sperelakis N. Bers D.M. J. Biol. Chem. 1998; 273: 10223-10231Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). As shown in Fig. 2A, WT mitochondria still exhibited high Ca2+-induced changes in light scatter in the presence of Ru360, at a concentration that was found to prevent the uptake of extramitochondrial Ca2+ (Fig. 1F, trace e). The lack of effect of Ru360 was also observed in the presence of cys A (Fig. 2A), or when the effect of Ca2+ was compared in WT versus cypD-KO mitochondria (Fig. 2B). To depolarize mitochondria completely, 1 μm SF 6847 was added to the medium, and the effects of Ca2+ and Ru360 were recorded. As shown in Fig. 2C, the presence of the uncoupler failed to provide extra protection against high Ca2+-induced swelling. Furthermore, the presence of the uncoupler negated the protective effects of substrates in WT mitochondria (Fig. 2D). Maximum swelling rates pooled from all experiments (expressed as in Fig. 1) for each condition and after each Ca2+ pulse are shown in Fig. 2E. The failure of Ru360 to protect against the Ca2+-induced large changes in light scatter shown in Fig. 2, A and B, could be explained by assuming that Ca2+ acted on the extramitochondrial side. To provide further evidence for this, we loaded isolated mitochondria with fura-2 and imaged them under wide field epifluorescence (Fig. 3A). This experimental setup (i) benefits from the spatial resolution in fura-2 imaging, avoiding a “contaminant” signal of leaked fura-2 in the extramitochondrial space and (ii) provides a valid quantitative signal of matrix [Ca2+] in the submicromolar range. Surprisingly, isolated mitochondria perfused with a buffer containing 0.1 mm CaCl2 showed robust increases in matrix-entrapped fura-2 fluorescence that exhibited only a partial sensitivity to Ru360 (10 μm) and Ruthenium red (10 μm) (Fig. 3B), arguing against the assumption that Ca2+ was acting exclusively on an extramitochondrial site when inducing changes in light scatter. To address the contribution of respiratory chain components to the protective effect of substrates against the Ca2+-induced changes in light scatter, we pretreated mitochondria with complex I (rotenone or piericidin A), complex III (myxothiazol or stigmatell" @default.
• W2061460906 created "2016-06-24" @default.
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• W2061460906 date "2011-02-01" @default.
• W2061460906 modified "2023-10-18" @default.
• W2061460906 title "Complex Contribution of Cyclophilin D to Ca2+-induced Permeability Transition in Brain Mitochondria, with Relation to the Bioenergetic State" @default.
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