Synaptic Mitochondria Are More Susceptible to Ca2+Overload than Nonsynaptic Mitochondria
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m510303200
ISSN1083-351X
AutoresMaile R. Brown, Patrick G. Sullivan, James W. Geddes,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoMitochondria in nerve terminals are subjected to extensive Ca2+fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca 2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders. Mitochondria in nerve terminals are subjected to extensive Ca2+fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca 2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders. Mitochondria are important regulators of cellular Ca2+ homeostasis, producers of ATP via oxidative phosphorylation, and regulators of cell death pathways (for reviews see Refs. 1Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1068) Google Scholar and 2Sullivan P.G. Rabchevsky A.G. Waldmeier P.C. Springer J.E. J. Neurosci. Res. 2005; 79: 231-239Crossref PubMed Scopus (321) Google Scholar). Mitochondria assist in maintaining Ca2+ homeostasis by sequestering and releasing Ca2+ (2Sullivan P.G. Rabchevsky A.G. Waldmeier P.C. Springer J.E. J. Neurosci. Res. 2005; 79: 231-239Crossref PubMed Scopus (321) Google Scholar, 3Bernardi P. Physiol. 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When the mitochondria become overloaded with Ca2+, they undergo the cataclysmic mitochondrial permeability transition (mPT) 3The abbreviations used are: mPT, mitochondrial permeability transition; BSA, bovine serum albumin; CaG5N, Ca2+ Green-5N hexapotassium salt; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CNS, central nervous system; COXIV, cytochrome oxidase subunit IV; CsA, cyclosporin A; DCF, 2′-7′-dichlorodihydro-fluorescein diacetate; F344, Fisher 344 rats; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; PSD-95, post-synaptic density 95 protein; ROS, reactive oxygen species; RuRed, Ruthenium Red; SD, Sprague-Dawley rats; TMRE, tetramethylrhodamine, ethyl ester perchlorate; TTBS, Tris-buffered saline containing 0.05% Tween 20; VDAC, voltage-dependent anion channel. via formation of a nonselective pore that allows solutes of 1500 daltons or smaller to pass through the usually impermeable inner mitochondrial membrane with a resultant rupture of the outer mitochondrial membrane caused by osmotic swelling (2Sullivan P.G. 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The heavy synaptic fractions exhibited a greater protein/lipid ratio, greater lipid peroxidation, and lower levels and activities of respiratory enzymes and were thought to reflect old mitochondria (34Battino M. Ferreiro M.S. Littarru G. Quiles J.L. Ramirez-Tortosa M.C. Huertas J.R. Mataix J. Villa R.E. Gorini A. Free Radic. Res. 2002; 36: 479-484Crossref PubMed Scopus (26) Google Scholar, 35Battino M. Quiles J.L. Huertas J.R. Mataix J.F. Villa R.F. Gorini A. J. Bioenerg. Biomembr. 2000; 32: 163-173Crossref PubMed Scopus (20) Google Scholar). In contrast, light synaptic and nonsynaptic mitochondria were largely similar in terms of enzyme, activities, lipid content, and lipid peroxidation. Percoll density gradients result in less contamination of the mitochondrial and synaptosomal fractions, and mitochondria isolated from synaptic and nonsynaptic populations exhibit similar enrichment, enzyme activity, and respiratory activity (32Rendon A. Masmoudi A. J. Neurosci. Methods. 1985; 14: 41-51Crossref PubMed Scopus (41) Google Scholar, 36Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (346) Google Scholar). The purpose of the present study was to compare the ability of well coupled isolated synaptic versus nonsynaptic brain mitochondria to accumulate exogenously added Ca2+. Reagents—Mannitol, sucrose, bovine serum albumin (BSA), EGTA, HEPES potassium salt, potassium phosphate monobasic anhydrous (KH2PO4), MgCl2, malate, pyruvate, ADP, oligomycin A, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Ca 2+ chloride, succinate, and Ruthenium Red (RuRed) were purchased from Sigma-Aldrich. A bicinchoninic acid protein assay kit and the Supersignal West Pico chemiluminescent substrate were purchased from Pierce. Percoll was purchased from Amersham Biosciences. Horseradish peroxidase-conjugated goat anti-mouse and antirabbit IgG secondary antibodies were purchased from Zymed Laboratories Inc. (San Francisco, CA). Tetramethylrhodamine, ethyl ester perchlorate (TMRE), Ca 2+ Green-5N hexapotassium salt (CaG5N), and 2′-7′-dichlorodihydro-fluorescein diacetate (DCF) were purchased from Molecular Probes (Eugene, OR). Mitochondrial Isolation—All experimental protocols involving animals were approved by the University of Kentucky Animal Use and Care Committee. Male Sprague-Dawley (SD) rats (250–300 g, 3 months of age) were used in all studies with the exception of the studies comparing the findings with those obtained in 3-month-old Fisher 344 (F344) rats. All of the animals were obtained from Harlan (Indianapolis, IN). As previously described (37Brown M.R. Sullivan P.G. Dorenbos K.A. Modafferi E.A. Geddes J.W. Steward O. J. Neurosci. Methods. 2004; 137: 299-303Crossref PubMed Scopus (88) Google Scholar), following carbon dioxide asphyxiation, the rats were decapitated, and the brains were rapidly removed. The cortices were dissected out and placed in a glass Dounce homogenizer containing five times the volume of isolation buffer (215 mm mannitol, 75 mm sucrose, 0.1% BSA, 20 mm HEPES, 1 mm EGTA, pH adjusted to 7.2 with KOH). The tissue was homogenized, and an equal volume of 30% Percoll in isolation buffer was added (∼4 ml). The resultant homogenate was layered on a discontinuous Percoll gradient with the bottom layer containing 40% Percoll solution in isolation buffer, followed by a 24% Percoll solution, and finally the sample in a 15% Percoll solution. The density gradients were spun in a Sorvall RC-5C plus superspeed refrigerated centrifuge (Asheville, NC) in a fixed angle SE-12 rotor at 30,400 × g for 10 min. Usage of two Percoll density gradients for cortical regions from each animal improved the resolution of nonsynaptic mitochondria on the Percoll density gradient. Following centrifugation, band 2 (synaptosomes) and band 3 (nonsynaptic mitochondria) (36Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (346) Google Scholar) were separately removed from the density gradient. Each fraction was placed in separate tubes, and 10 ml of isolation buffer was added. The samples were washed by centrifugation at 16,700 × g for 15 min. The supernatant was discarded, and the loose pellet was resuspended in the 1 ml of isolation buffer. A nitrogen cell disruption bomb (model 4639; Parr Instrument Company, Moline, IL) cooled to 4 °C was used to burst the synaptosomes within this fraction (37Brown M.R. Sullivan P.G. Dorenbos K.A. Modafferi E.A. Geddes J.W. Steward O. J. Neurosci. Methods. 2004; 137: 299-303Crossref PubMed Scopus (88) Google Scholar, 38Sullivan P.G. Dragicevic N.B. Deng J.-H. Bai Y. Dimayuga E. Ding Q. Chen Q. Bruce-Keller A.J. Keller J.N. J. Biol. Chem. 2004; : M313579200Google Scholar). Both the nonsynaptic mitochondria and synaptosomes were placed in the nitrogen disruption bomb for 10 min at 1000 p.s.i. Previously, we demonstrated that the nitrogen disruption method does not impair mitochondrial function (37Brown M.R. Sullivan P.G. Dorenbos K.A. Modafferi E.A. Geddes J.W. Steward O. J. Neurosci. Methods. 2004; 137: 299-303Crossref PubMed Scopus (88) Google Scholar). The nonsynaptic mitochondrial and the nitrogen-disrupted synaptosomal mitochondrial fractions were placed in separate 15-ml conical tubes. An equal volume of 30% Percoll was added to each sample, and discontinuous Percoll density gradient centrifugation was performed as described above. Band 3 was obtained from each of the gradients, and 10 ml of isolation buffer without EGTA (215 mm mannitol, 75 mm sucrose, 0.1% BSA, 20 mm HEPES, pH is adjusted to 7.2 with KOH) was added. The fractions were centrifuged at 16,700 × g for 15 min and subsequently at 11,000 × g for 10 min. The resultant pellet was resuspended in 1 ml of isolation buffer without EGTA and centrifuged at 10,000 × g for 10 min. The final mitochondrial pellet was resuspended in isolation buffer without EGTA to yield a protein concentration of ∼10 mg protein/ml and stored on ice. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce). Respiration Measurements—The respiratory activity of isolated mitochondria was measured using a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) as previously described (39Sullivan P.G. Thompson M.B. Scheff S.W. Exp. Neurol. 1999; 160: 226-234Crossref PubMed Scopus (291) Google Scholar). Approximately 100 μg protein/ml of isolated nonsynaptic or synaptic mitochondria were suspended in a sealed, constantly stirred, and thermostatically controlled chamber at 37 °C in KCl respiration buffer (125 mm KCl, 0.1% BSA, 20 mm HEPES, 2 mm MgCl2, 2.5 mm KH2PO4, pH 7.2). The rate of oxygen consumption was calculated based on the slope of the response of isolated mitochondria to the successive administration of oxidative substrates (5 mm pyruvate and 2.5 mm malate): 150 μm ADP added twice in 1-min intervals; 1 μm oligomycin; 1 μm FCCP; and finally 1 mm succinate (40Sullivan P.G. Dube C. Dorenbos K. Steward O. Baram T.Z. Ann. Neurol. 2003; 53: 711-717Crossref PubMed Scopus (217) Google Scholar). The respiratory control ratio was determined by dividing the rate of oxygen consumption/min for state III (in the presence of ADP, second addition) by state IV (in the absence of ADP and presence of oligomycin). Only isolated mitochondrial preparations with an respiratory control ratio of over 5 were used in the study. The states of mitochondrial respiration described by Chance and Williams (41Chance B. Williams G.R. J. Biol. Chem. 1955; 217: 383-393Abstract Full Text PDF PubMed Google Scholar) were also calculated (nmol of oxygen consumed/mg of protein) in KCl respiration buffer. Fluorescent Spectrofluorophotometer Assays—Fractions enriched in nonsynaptic and synaptic mitochondria (50 μg protein/ml) were placed in 2 ml of KCl respiration buffer in a constantly stirred, temperature-controlled cuvette at 37 °C with 100 nm CaG5N; excitation, 506 nm; emission, 532 nm; and 100 nm TMRE; excitation, 550 nm; emission, 575 nm; in the Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan). CaG5N was used to monitor extramitochondrial Ca 2+, and TMRE was used to simultaneously monitor changes in Δψm. Each time scan began with a base-line reading followed by a 5 mm pyruvate and 2.5 mm malate addition at 1 min, then 150 μm ADP at 2 min, and then 1 μm oligomycin at 3 min. At 5 min, Ca2+ was added by a gradual delivery via an KD Scientific model 310 series infusion syringe pump (Holliston, MA) (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 42Zoccarato F. Nicholls D. Eur. J. Biochem. 1982; 127: 333-338Crossref PubMed Scopus (107) Google Scholar) (160 nmol of Ca2+/mg of protein/min) or through bolus additions (1000 nmol of Ca2+/mg of protein or 50 μm Ca 2+) until the mitochondria were no longer able to buffer the added Ca 2+. The chemical uncoupler CCCP was added toward the end of each run. The traces presented are representative of at least three separate, independent experiments. The spectrofluorophotometer traces were quantified by calculating the average base-line CaG5N fluorescence readings 1 min prior to the beginning of the Ca 2+ infusion or before the first bolus addition using the Shimadzu Hyper RF software and Microsoft Excel. The time point at which the CaG5N signal was 150% above the average base-line reading was considered to be the point at which the mitochondria were overloaded and no longer capable of removing Ca 2+ from the media. Mitochondrial Ca 2+ uptake capacity was calculated as the amount of Ca2+ added or infused (nmol/mg) prior to the point at which the CaG5N signal was 150% above the average base-line reading. Reactive Oxygen Species Production—Mitochondrial ROS production was measured using 25 μm DCF (485 nm, 530 nm) in the Biotek Synergy HT plate reader as previously described (17Brown M.R. Geddes J.W. Sullivan P.G. J. Bioenerg. Biomembr. 2004; 36: 401-406Crossref PubMed Scopus (75) Google Scholar, 43Sullivan P.G. Rabchevsky A.G. Keller J.N. Lovell M. Sodhi A. Hart R.P. Scheff S.W. J. Comp. Neurol. 2004; 474: 524-534Crossref PubMed Scopus (101) Google Scholar, 44Sullivan P.G. Geiger J.D. Mattson M.P. Scheff S.W. Ann. Neurol. 2000; 48: 723-729Crossref PubMed Scopus (235) Google Scholar). Isolated mitochondria (25 μg of protein/ml) were added to 100 μl of KCl respiration buffer with 5 mm pyruvate and 2.5 mm malate as oxidative substrates at 37 °C. ROS production was calculated as the maximum DCF fluorescence following 15 min of incubation, expressed in arbitrary fluorescence units. Mitochondrial ROS production in the presence of oligomycin (to induce maximal ROS production) or FCCP (to induce minimal ROS production) was also determined to ensure that our measurements were within the range of the ROS indicator. Western Blotting—Isolated nonsynaptic and synaptic mitochondria in isolation buffer plus a protease inhibitor mixture (Complete Mini; Roche Applied Science) were centrifuged at 10,000 × g for 10 min. The resultant mitochondrial pellet was resuspended in 100 μl of isolation buffer plus protease inhibitors with 0.01% Triton X-100, sonicated for 20 s, and centrifuged at 10,000 × g for 10 min. The supernatant was used for Western blots. Sample buffer was added to the samples based on relative protein concentrations determined from the bicinchoninic acid protein assay, and all of the lanes were loaded with the same amount of protein (5 μg/lane). The samples were separated by SDS-PAGE using 10 or 12.5% Trisacrylamide/bis gels, along with Bio-Rad low range molecular weight markers. Following SDS-PAGE, the polypeptides were transferred electrophoretically onto 0.2 μm nitrocellulose membranes. The membranes were incubated at room temperature for 1 h in 5% nonfat milk in 50 mm Tris-saline containing 0.05% Tween 20 at pH 7.5 (TTBS). The blots were incubated overnight in the primary antibody in TTBS at 22 °C. The primary antibodies used in study included monoclonal cytochrome oxidase subunit IV (COXIV) at 1:20,000 (Molecular Probes); monoclonal post-synaptic density 95 protein (PSD-95) at 1:20,000 (BD Biosciences, San Jose, CA); and polyclonal voltage-dependent anion channel (VDAC) at 1:10,000 (Affinity Bioreagents, Golden, CO). After overnight incubation in primary antibody, the membranes were rinsed three times in TTBS and incubated in secondary antibody for 1 h either in horseradish peroxidase-conjugated goat anti-mouse IgG (1:3000) for COXIV and PSD-95 or in horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000) for VDAC. The blots were rinsed thoroughly in TTBS and were briefly incubated in the Pierce SuperSignal Pico chemiluminescent substrate. Finally, the blots were visualized using a Kodak Image Station 2000R and the Kodak Molecular Imaging software. Statistics—Statistical analyses were performed using either an unpaired t test or a one-way analysis of variance (p < 0.05) with Scheffé's post hoc analysis when appropriate. The results are expressed as the group means (± S.E.) from at least three independent experiments, and group size is indicated for each experiment in the figure legends. Isolation of Synaptic Mitochondria—To isolate well coupled synaptic mitochondria, the isolation procedure utilized two separate centrifugations on discontinuous Percoll gradients and a nitrogen decompression technique (Fig. 1). Both the synaptic and nonsynaptic mitochondria underwent the nitrogen disruption and two runs through discontinuous Percoll density gradients. The nitrogen cell disruption avoids the damage to mitochondria caused by detergent-based disruption methods (45Brustovetsky N. Jemmerson R. Dubinsky J.M. Neurosci. Lett. 2002; 332: 91-94Crossref PubMed Scopus (20) Google Scholar) and has been demonstrated to yield well coupled mitochondria (37Brown M.R. Sullivan P.G. Dorenbos K.A. Modafferi E.A. Geddes J.W. Steward O. J. Neurosci. Methods. 2004; 137: 299-303Crossref PubMed Scopus (88) Google Scholar). The average mitochondrial yields from cortical tissue pooled from two rats were 946 μg for the nonsynaptic fraction and 453 μg for the synaptic fraction. Western blots were performed using antibodies to probe for the outer mitochondrial membrane protein, VDAC; the inner mitochondrial membrane protein, COXIV; and the synaptosomal protein, PSD-95 (Fig. 2). The nonsynaptic mitochondrial fraction had strong immunoreactivity for the mitochondrial membrane proteins COXIV and VDAC but not for PSD-95. The synaptosomes were positive for PSD-95 marker after the first Percoll centrifugation and after the nitrogen disruption. Following the second Percoll gradient, the synaptic mitochondrial-enriched fraction demonstrated strong immunoreactivity for both mitochondrial markers, but PSD-95 was not detected.FIGURE 2Mitochondrial and synaptosomal protein profiles of fractions throughout the isolation procedure. The mitochondrial markers COXIV and VDAC became enriched after separation on the Percoll density gradient and subsequent washing. The synaptosomes taken after the first Percoll density gradient and after the nitrogen cell disruption were positive for the synaptosomal marker PSD-95. After the nitrogen-disrupted synaptosomes were run through the second Percoll gradient, the mitochondrial markers increased in intensity (final two lanes), indicating similar enrichment of mitochondria in both the synaptic and nonsynaptic mitochondrial preparations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Similar Bioenergetics in Nonsynaptic and Synaptic Mitochondria—Following mitochondrial isolation, the rate of oxygen consumption in presence of pyruvate and malate as the oxidative substrates was measured (Fig. 3). Similar respiration rates were observed in the nonsynaptic and synaptic mitochondria (Fig. 3A). There was no significant difference in oxygen consumption in any of the different classical states of respiration (Fig. 3B)or in the respiratory control ratio (Fig. 3C). Together, the results demonstrate that both populations of isolated mitochondria were well coupled and bioenergetically active following the isolation procedure. Increased Ca 2+ Accumulation in Nonsynaptic versus Synaptic Mitochondria in Two Rat Strains—Isolated nonsynaptic and synaptic mitochondria were incubated with an extramitochondrial, low affinity Ca2+ fluorescent dye, CaG5N, and a Δψm fluorescent indicator, TMRE, and placed in a constantly stirred, temperature-controlled cuvette at 37 °C in a spectrofluorophotometer. All of the functional assays on the mitochondria were done in a KCl-based respiration buffer containing magnesium, inorganic phosphates, and BSA. The indicator TMRE was used in "quench" mode such that at high mitochondrial Δψm fluorescence is lower than at lower Δψm, because of dye stacking within the matrix (46Drummond R.M. Mix T.C. Tuft R.A. Walsh Jr., J.V. Fay F.S. J. Physiol. 2000; 522: 375-390Crossref PubMed Scopus (57) Google Scholar). After obtaining a base-line reading, oxidative substrates (5 mm pyruvate and 2.5 mm malate) were added, allowing the mitochondria to generate a high Δψm indicated by the sharp downward deflection of the TMRE trace (Fig. 4C). One min later, 150μm ADP was added, which caused the mitochondria to depolarize and use their Δψm to phosphorylate the added ADP to ATP. Afterward, the ATP synthase inhibitor oligomycin (1 μm) was added, and the high Δψm was reestablished. These early additions served as internal controls in each experiment to ensure that
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