SynapticMitochondriaAreMoreSusceptibletoCa2 ...11658 JOURNALOFBIOLOGICALCHEMISTRY VOLUME281•NUMBER17•APRIL28,2006. enzyme,activities,lipidcontent,andlipidperoxidation.Percolldensity

Synaptic Mitochondria Are More Susceptible to Ca2�

Overload than Nonsynaptic Mitochondria*

Received for publication, September 20, 2005, and in revised form, January 25, 2006 Published, JBC Papers in Press, March 3, 2006, DOI 10.1074/jbc.M510303200

Maile R. Brown‡§¶1, Patrick G. Sullivan¶�, and James W. Geddes§¶�2

From the ‡Graduate Center for Gerontology, §Sanders-Brown Center on Aging, ¶Spinal Cord and Brain Injury Research Center,and the �Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536

Mitochondria in nerve terminals are subjected to extensive Ca2�

fluxes and high energy demands, but the extent to which the synap-tic mitochondria buffer Ca2� is unclear. In this study, we identifieda difference in the Ca2� clearance ability of nonsynaptic versus syn-aptic mitochondrial populations enriched from rat cerebral cortex.Mitochondria were isolated using Percoll discontinuous gradientsin combination with high pressure nitrogen cell disruption. Mito-chondria in the nonsynaptic fraction originate from neurons andother cell types including glia, whereasmitochondria enriched froma synaptosomal fraction are predominantly neuronal and presynap-tic 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 mito-chondria were able to accumulate significantly more exogenouslyadded Ca2� than the synaptic mitochondria before undergoing mito-chondrial permeability transition, observed as a loss in mitochondrialmembranepotential anddecreasedCa2�uptake.The limited ability ofsynaptic mitochondria to accumulate Ca2� could result from severalfactors including aprimary functionofATPproduction to support thehighenergydemandofpresynaptic terminals, their relative isolation incomparison with the threads or clusters of mitochondria found in thesoma of neurons and glia, or the older age and increased exposure tooxidative damage of synaptic versus nonsynaptic mitochondria. Bymore readily undergoing permeability transition, synaptic mitochon-dria may initiate neuron death in response to insults that elevate syn-aptic levels of intracellular Ca2�, consistent with the early degenera-tion of distal axon segments in neurodegenerative disorders.

Mitochondria are important regulators of cellular Ca2� homeostasis,producers of ATP via oxidative phosphorylation, and regulators of celldeathpathways (for reviews seeRefs. 1 and2).Mitochondria assist inmain-taining Ca2� homeostasis by sequestering and releasing Ca2� (2–4). Nor-mal Ca2� cycling occurs by the movement of Ca2� into mitochondria viatheCa2�uniporter and slowefflux via theNa�/Ca2� antiporter or byNa�-independent mechanisms (1, 3). Isolated mitochondria in the presence ofphosphatetakeupCa2� toafixedcapacity, inamembranepotential (��m)-dependent fashion (5–7).WhenthemitochondriabecomeoverloadedwithCa2�, they undergo the cataclysmic mitochondrial permeability transition

(mPT)3 via formation of a nonselective pore that allows solutes of 1500daltons or smaller to pass through the usually impermeable inner mito-chondrial membrane with a resultant rupture of the outer mitochondrialmembrane caused by osmotic swelling (2, 8–12).Previous studies have demonstrated substantial mitochondrial heter-

ogeneity that exists among organs and within the CNS. Nonsynapticbrain mitochondria are more resistant to Ca2�-induced opening ofmPT, assessed by mitochondrial swelling, when compared with livermitochondria (13–15). Within the CNS, there are regional differences inmitochondrial populations with regard to Ca2�-induced mPT thresholdand reactive oxygen species (ROS) production (16–18). There is alsoregional and cellular heterogeneity in the composition, morphology,and trafficking of mitochondria (19).Synaptic mitochondria exist in an environment where they are

exposed to extensive Ca2� influx. Although synaptic mitochondriaare important in Ca2� clearance, it is uncertain whether this is pre-dominantly by direct Ca2� removal or by providing ATP for theplasma membrane Na�/Ca2� exchanger (20). Presynaptic mito-chondria are typically punctuate and isolated, in contrast to themitochondrial threads and clusters found in other regions of neu-rons and astrocytes (21, 22). Synaptic mitochondria are synthesizedin the cell body of neurons and then transported down the axon ordendrite (23–25). Within the central nervous system, mitochondriahave an apparent half-life of approximately one month (26). As aresult of transport, synaptic mitochondria may be “older” than mito-chondria in the soma of neurons and glial cells and may exhibitgreater cumulative damage from oxidative stress. As mitochondriaage, they become more heterogeneous and on average become moredepolarized (27). Because of their different function, morphology, orage, presynaptic mitochondria may handle Ca2� differently thanmitochondria in other regions of neurons and other cell types.Previous studies have compared isolated nonsynaptic and synaptic

mitochondria with regard to metabolism and lipid composition (19,28–35). Distinctions in Ca2� handling between isolated synaptic andnonsynaptic mitochondria have not been examined previously. In stud-ies using Ficoll density gradients, mitochondria were isolated from non-synaptic, “light” synaptic, and “heavy” synaptic fractions. The heavy syn-aptic fractions exhibited a greater protein/lipid ratio, greater lipidperoxidation, and lower levels and activities of respiratory enzymes andwere thought to reflect old mitochondria (34, 35). In contrast, lightsynaptic and nonsynaptic mitochondria were largely similar in terms of

* This work was supported by National Institutes of Health, United States Public HealthService Grants AG10836 and NS045726 (to J. W. G.) and NS048191 and NS046426 (toP. G. S.). The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Predoctoral trainee under National Institutes of Health Training Grant AG00264.Present address: Dept. of Pharmacology, Yale University School of Medicine, NewHaven, CT 06520.

2 To whom correspondence should be addressed: B379 BBSRB, University of Ken-tucky, Lexington, KY 40536-0509. Tel.: 859-323-5135; Fax: 859-257-5737; E-mail:[email protected].

3 The abbreviations used are: mPT, mitochondrial permeability transition; BSA, bovineserum albumin; CaG5N, Ca2� Green-5N hexapotassium salt; CCCP, carbonyl cyanide3-chlorophenylhydrazone; CNS, central nervous system; COXIV, cytochrome oxidasesubunit 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 esterperchlorate; TTBS, Tris-buffered saline containing 0.05% Tween 20; VDAC, voltage-dependent anion channel.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 17, pp. 11658 –11668, April 28, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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enzyme, activities, lipid content, and lipid peroxidation. Percoll densitygradients result in less contamination of the mitochondrial and synap-tosomal fractions, and mitochondria isolated from synaptic and non-synaptic populations exhibit similar enrichment, enzyme activity, andrespiratory activity (32, 36). The purpose of the present study was tocompare the ability of well coupled isolated synaptic versus nonsynapticbrain mitochondria to accumulate exogenously added Ca2�.

MATERIALS AND METHODS

Reagents—Mannitol, sucrose, bovine serum albumin (BSA), EGTA,HEPES potassium salt, potassium phosphate monobasic anhydrous(KH2PO4),MgCl2, malate, pyruvate, ADP, oligomycin A, carbonyl cyanide4-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanide 3-chlo-rophenylhydrazone (CCCP),Ca2� chloride, succinate, andRutheniumRed(RuRed)werepurchased fromSigma-Aldrich.Abicinchoninic acidproteinassay kit and the Supersignal West Pico chemiluminescent substrate werepurchased from Pierce. Percoll was purchased from Amersham Bio-sciences. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit IgG secondary antibodieswere purchased fromZymedLaboratoriesInc. (San Francisco, CA). Tetramethylrhodamine, ethyl ester perchlorate(TMRE), Ca2� Green-5N hexapotassium salt (CaG5N), and 2�-7�-di-chlorodihydro-fluorescein diacetate (DCF) were purchased fromMolecular Probes (Eugene, OR).

Mitochondrial Isolation—All experimental protocols involving ani-malswere approved by theUniversity of KentuckyAnimalUse andCareCommittee. Male Sprague-Dawley (SD) rats (250–300 g, 3 months ofage) were used in all studies with the exception of the studies comparingthe findings with those obtained in 3-month-old Fisher 344 (F344) rats.All of the animals were obtained from Harlan (Indianapolis, IN). Aspreviously described (37), following carbon dioxide asphyxiation, therats were decapitated, and the brains were rapidly removed. The corti-ces were dissected out and placed in a glass Dounce homogenizer con-taining five times the volume of isolation buffer (215 mM mannitol, 75mM sucrose, 0.1% BSA, 20 mMHEPES, 1 mM EGTA, pH adjusted to 7.2with KOH). The tissue was homogenized, and an equal volume of 30%Percoll in isolation buffer was added (�4 ml). The resultant homoge-nate was layered on a discontinuous Percoll gradient with the bottomlayer containing 40% Percoll solution in isolation buffer, followed by a24% Percoll solution, and finally the sample in a 15% Percoll solution.The density gradients were spun in a Sorvall RC-5C plus superspeedrefrigerated centrifuge (Asheville, NC) in a fixed angle SE-12 rotor at30,400� g for 10min. Usage of two Percoll density gradients for corticalregions from each animal improved the resolution of nonsynapticmito-chondria on the Percoll density gradient.Following centrifugation, band 2 (synaptosomes) and band 3 (non-

synaptic mitochondria) (36) were separately removed from the densitygradient. Each fraction was placed in separate tubes, and 10 ml of isola-tion buffer was added. The samples were washed by centrifugation at16,700 � g for 15 min. The supernatant was discarded, and the loosepellet was resuspended in the 1 ml of isolation buffer. A nitrogen celldisruption bomb (model 4639; Parr Instrument Company, Moline, IL)cooled to 4 °C was used to burst the synaptosomes within this fraction(37, 38). Both the nonsynaptic mitochondria and synaptosomes wereplaced in the nitrogen disruption bomb for 10 min at 1000 p.s.i. Previ-ously, we demonstrated that the nitrogen disruption method does notimpair mitochondrial function (37).The nonsynaptic mitochondrial and the nitrogen-disrupted synapto-

somal mitochondrial fractions were placed in separate 15-ml conicaltubes. An equal volume of 30% Percoll was added to each sample, anddiscontinuous Percoll density gradient centrifugation was performed as

described above. Band 3was obtained from each of the gradients, and 10ml of isolation buffer without EGTA (215mMmannitol, 75mM 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 subse-quently at 11,000 � g for 10 min. The resultant pellet was resuspendedin 1 ml of isolation buffer without EGTA and centrifuged at 10,000 � gfor 10 min. The final mitochondrial pellet was resuspended in isolationbuffer without EGTA to yield a protein concentration of �10 mg pro-tein/ml and stored on ice. Protein concentration was determined usingthe bicinchoninic acid protein assay (Pierce).

Respiration Measurements—The respiratory activity of isolated mito-chondria was measured using a Clark-type oxygen electrode (HansatechInstruments,Norfolk,UK) aspreviouslydescribed (39).Approximately 100�g protein/ml of isolated nonsynaptic or synaptic mitochondria were sus-pended in a sealed, constantly stirred, and thermostatically controlledchamber at 37 °C in KCl respiration buffer (125mMKCl, 0.1% BSA, 20mM

HEPES, 2 mM MgCl2, 2.5 mM KH2PO4, pH 7.2). The rate of oxygenconsumption was calculated based on the slope of the response of iso-lated mitochondria to the successive administration of oxidative sub-strates (5mM pyruvate and 2.5mMmalate): 150�MADP added twice in1-min intervals; 1 �M oligomycin; 1 �M FCCP; and finally 1 mM succi-nate (40). The respiratory control ratio was determined by dividing therate of oxygen consumption/min for state III (in the presence of ADP,second addition) by state IV (in the absence of ADP and presence ofoligomycin). Only isolated mitochondrial preparations with an respira-tory control ratio of over 5 were used in the study. The states of mito-chondrial respiration described by Chance andWilliams (41) were alsocalculated (nmol of oxygen consumed/mg of protein) in KCl respirationbuffer.

Fluorescent Spectrofluorophotometer Assays—Fractions enriched innonsynaptic and synaptic mitochondria (50 �g protein/ml) wereplaced in 2 ml of KCl respiration buffer in a constantly stirred, tem-perature-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 spectrofluoropho-tometer (Kyoto, Japan). CaG5Nwas used to monitor extramitochon-drial Ca2�, and TMRE was used to simultaneously monitor changesin ��m. Each time scan began with a base-line reading followed by a5 mM pyruvate and 2.5 mM malate addition at 1 min, then 150 �M

ADP at 2min, and then 1 �M oligomycin at 3 min. At 5min, Ca2� wasadded by a gradual delivery via an KD Scientific model 310 seriesinfusion syringe pump (Holliston, MA) (5, 42) (160 nmol of Ca2�/mgof protein/min) or through bolus additions (1000 nmol of Ca2�/mgof protein or 50 �MCa2�) until the mitochondria were no longer ableto buffer the added Ca2�. The chemical uncoupler CCCP was addedtoward the end of each run. The traces presented are representativeof at least three separate, independent experiments.The spectrofluorophotometer traces were quantified by calculating

the average base-line CaG5N fluorescence readings 1 min prior to thebeginning of the Ca2� infusion or before the first bolus addition usingthe ShimadzuHyper RF software andMicrosoft Excel. The time point atwhich the CaG5N signal was 150% above the average base-line readingwas considered to be the point at which the mitochondria were over-loaded and no longer capable of removing Ca2� from the media. Mito-chondrial Ca2� uptake capacity was calculated as the amount of Ca2�

added or infused (nmol/mg) prior to the point at which the CaG5N signalwas 150% above the average base-line reading.

Reactive Oxygen Species Production—Mitochondrial ROS produc-tion was measured using 25 �M DCF (485 nm, 530 nm) in the BiotekSynergy HT plate reader as previously described (17, 43, 44). Isolated

Ca2� Buffering Differences in Brain Mitochondria

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mitochondria (25 �g of protein/ml) were added to 100 �l of KCl respi-ration buffer with 5 mM pyruvate and 2.5 mM malate as oxidative sub-strates at 37 °C. ROS production was calculated as the maximum DCFfluorescence following 15 min of incubation, expressed in arbitrary flu-orescence units. Mitochondrial ROS production in the presence of oli-gomycin (to inducemaximal ROS production) or FCCP (to inducemin-imal ROS production) was also determined to ensure that ourmeasurements were within the range of the ROS indicator.

Western Blotting—Isolated nonsynaptic and synaptic mitochondria inisolation buffer plus a protease inhibitor mixture (Complete Mini; RocheApplied Science) were centrifuged at 10,000 � g for 10 min. The resultantmitochondrial pellet was resuspended in 100 �l of isolation buffer plusprotease inhibitorswith 0.01%TritonX-100, sonicated for 20 s, and centri-fuged at 10,000 � g for 10 min. The supernatant was used for Western

blots. Sample buffer was added to the samples based on relative proteinconcentrations determined from the bicinchoninic acid protein assay, andall of the lanes were loaded with the same amount of protein (5 �g/lane).The samples were separated by SDS-PAGE using 10 or 12.5% Tris-

acrylamide/bis gels, along with Bio-Rad low range molecular weightmarkers. Following SDS-PAGE, the polypeptides were transferred elec-trophoretically onto 0.2�Mnitrocellulosemembranes. Themembraneswere incubated at room temperature for 1 h in 5% nonfat milk in 50mM

Tris-saline containing 0.05% Tween 20 at pH 7.5 (TTBS). The blotswere incubated overnight in the primary antibody inTTBS at 22 °C. Theprimary antibodies used in study includedmonoclonal cytochrome oxi-dase subunit IV (COXIV) at 1:20,000 (Molecular Probes); monoclonalpost-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 overnightincubation in primary antibody, themembraneswere rinsed three timesin TTBS and incubated in secondary antibody for 1 h either in horse-radish peroxidase-conjugated goat anti-mouse IgG (1:3000) for COXIVand PSD-95 or in horseradish peroxidase-conjugated goat anti-rabbitIgG (1:3000) for VDAC. The blots were rinsed thoroughly in TTBS andwere briefly incubated in the Pierce SuperSignal Pico chemiluminescentsubstrate. Finally, the blots were visualized using a Kodak Image Station2000R and the Kodak Molecular Imaging software.

Statistics—Statistical analyses were performed using either anunpaired t test or a one-way analysis of variance (p � 0.05) with Schef-fe’s post hoc analysis when appropriate. The results are expressed as thegroupmeans (� S.E.) from at least three independent experiments, andgroup size is indicated for each experiment in the figure legends.

RESULTS

Isolation of SynapticMitochondria—To isolate well coupled synapticmitochondria, the isolation procedure utilized two separate centrifuga-tions on discontinuous Percoll gradients and a nitrogen decompressiontechnique (Fig. 1). Both the synaptic and nonsynaptic mitochondriaunderwent the nitrogen disruption and two runs through discontinuousPercoll density gradients. The nitrogen cell disruption avoids the dam-age to mitochondria caused by detergent-based disruption methods(45) and has been demonstrated to yield well coupled mitochondria(37). The average mitochondrial yields from cortical tissue pooled fromtwo rats were 946 �g for the nonsynaptic fraction and 453 �g for thesynaptic fraction. Western blots were performed using antibodies toprobe for the outer mitochondrial membrane protein, VDAC; the innermitochondrial membrane protein, COXIV; and the synaptosomal pro-tein, PSD-95 (Fig. 2). The nonsynaptic mitochondrial fraction hadstrong immunoreactivity for the mitochondrial membrane proteins

FIGURE 1. Overview of isolation procedure for nonsynaptic and synaptic brainmitochondria.

FIGURE 2. Mitochondrial and synaptosomalprotein profiles of fractions throughout theisolation procedure. The mitochondrial markersCOXIV and VDAC became enriched after separa-tion on the Percoll density gradient and subse-quent washing. The synaptosomes taken after thefirst Percoll density gradient and after the nitrogencell disruption were positive for the synaptosomalmarker PSD-95. After the nitrogen-disrupted synap-tosomes were run through the second Percoll gradi-ent, the mitochondrial markers increased in inten-sity (final two lanes), indicating similar enrichment ofmitochondria in both the synaptic and nonsynapticmitochondrial preparations.




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COXIV and VDAC but not for PSD-95. The synaptosomes were posi-tive for PSD-95marker after the first Percoll centrifugation and after thenitrogen disruption. Following the second Percoll gradient, the synapticmitochondrial-enriched fraction demonstrated strong immunoreactiv-ity for both mitochondrial markers, but PSD-95 was not detected.

Similar Bioenergetics in Nonsynaptic and Synaptic Mitochondria—Fol-lowing mitochondrial isolation, the rate of oxygen consumption in pres-ence of pyruvate andmalate as the oxidative substrates wasmeasured (Fig.3). Similar respiration rates were observed in the nonsynaptic and synapticmitochondria (Fig. 3A). There was no significant difference in oxygen con-sumption in any of the different classical states of respiration (Fig. 3B) or inthe respiratory control ratio (Fig. 3C). Together, the results demonstratethat both populations of isolated mitochondria were well coupled andbioenergetically active following the isolation procedure.

Increased Ca2� Accumulation in Nonsynaptic versus Synaptic Mito-chondria in Two Rat Strains—Isolated nonsynaptic and synaptic mito-chondria were incubated with an extramitochondrial, low affinity Ca2�

fluorescent dye, CaG5N, and a ��m fluorescent indicator, TMRE, andplaced in a constantly stirred, temperature-controlled cuvette at 37 °C in aspectrofluorophotometer. All of the functional assays on themitochondriawere done in a KCl-based respiration buffer containing magnesium, inor-ganic phosphates, and BSA. The indicator TMRE was used in “quench”mode such that at high mitochondrial ��m fluorescence is lower than atlower��m, because of dye stackingwithin thematrix (46). After obtainingabase-line reading,oxidative substrates (5mMpyruvateand2.5mMmalate)were added, allowing the mitochondria to generate a high ��m indicatedby the sharp downward deflection of the TMRE trace (Fig. 4C). One minlater, 150�MADPwasadded,whichcaused themitochondria todepolarizeanduse their��mtophosphorylate theaddedADPtoATP.Afterward, theATP synthase inhibitor oligomycin (1 �m) was added, and the high ��mwas reestablished. These early additions served as internal controls in each

experiment to ensure that both the nonsynaptic and the synaptic mito-chondrial preparations were well coupled and bioenergetically competentfor subsequent experiments.Next, the ability of isolated nonsynaptic and synapticmitochondria to

buffer Ca2� was investigated using two approaches in two rat strains.First, Ca2�was infused at 160 nmol of Ca2�/mg of protein/min using aninfusion pump (Fig. 4) into the cuvette. The infusionwas terminated afterthe mitochondria were no longer able to accumulate the added Ca2� asdemonstrated by an increase in CaG5N signal. Two minutes later, CCCPwas added causing the total collapse of any remaining ��m and a totalrelease ofCa2�within themitochondria. To determinewhether the resultsobtainedwere specific to theSDrats (Fig. 4,AandB), anoutbred lineof rats,experiments were also performed using an inbred line, F344 rats (Fig. 4, CandD). Inbothrat strains, thenonsynapticmitochondrialpopulationswereable tobuffer significantlymoreof the infusedCa2� than the synapticmito-chondria. Also, in both strains, collapse of the ��m preceded the impair-ment of Ca2� accumulation.The second approach we utilized was bolus additions of 1000 nmol of

Ca2�/mg of protein (50 �M) to isolated mitochondria populations to sim-ulate pathologic loads ofCa2� (Fig. 5). Following the first additions ofCa2�,the CaG5N signal increased transiently. This was accompanied by a loss in��m, which was utilized to drive the uptake of Ca2� via the electrogenicCa2� uniporter. Both the CaG5N and TMRE fluorescence then returnedtoward base line as the mitochondria were able to take up the extramito-chondrialCa2� (Fig. 5).Aswithaconstant infusion, the synapticmitochon-drial populations were unable to buffer as much Ca2� before undergoingmPT as nonsynpaticmitochondria. The kinetics of Ca2� uptake in the twopopulations were similar with the first bolus addition, but with the secondbolus addition the rate of Ca2� uptake was decreased in synaptic versusnonsynaptic mitochondria, and this decrease was exacerbated with subse-quent additions of Ca2�. The loss of��mwas caused byCa2�, because the

FIGURE 3. Differences in the mitochondrial bio-energetics were not observed between nonsyn-aptic and synaptic mitochondria. A, representa-tive traces from nonsynaptic (darker traces) andsynaptic (lighter trace) mitochondrial oxygen con-sumption measurements in the presence of oxida-tive substrates (pyruvate and malate), ADP, oligomy-cin, FCCP, and succinate. B, there were no significantdifferences in any of the states of respiration. State IIIis particularly high because of the well coupledmitochondria produced through this isolationprocedure. C, the respiratory control ratios (RCR),which is state III respiration divided by state IV res-piration, were similar for both populations andwere well above the acceptable range of 5. Theresults are the means � S.E. from seven independ-ent experiments.




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addition of EGTA, the Ca2� chelator, was able to partially rescue the��m(Fig. 5B).Overall, there was a significant increase in the amount of Ca2� (nmol

of Ca2�/mg of protein) taken up prior to mPT by nonsynaptic mito-chondrial populations as compared with synaptic mitochondrial popu-lations isolated from SD and F344 brain tissue (Fig. 6B). When the mPTinhibitor cyclosporin A (CsA) was added, there was an increase in theamount of Ca2� accumulated prior to mPT by both nonsynaptic andsynaptic mitochondria (Fig. 6, A and C). However, the influence of CsAon Ca2� accumulation was significant (p � 0.005) in nonsynaptic mito-chondrial fractions but not in synaptic mitochondria. In the presence ofCsA, Ca2� accumulation prior to mPT by nonsynaptic mitochondriaremained greater than in synaptic mitochondria (p � 0.0001) (Fig. 6C).The greater uptake capacity of nonsynaptic mitochondria was also evi-dent using the Ca2� bolus paradigm (p � 0.004) (Fig. 6D).

To investigate the possibility that the initial Ca2� loading within themitochondria was different between the nonsynaptic and synapticmitochondria after the isolation technique, CCCP, a chemical uncou-pler, was added at 7 min after the addition of substrates to induce effluxof the stored Ca2� (Fig. 7). There was no difference in the level of the

CaG5N signal after CCCP addition, indicating no substantial differences inthe amount of Ca2� contained with the matrix after isolation. This arguesagainst differential Ca2� loading in synaptic versus nonsynapticmitochon-dria as a cause of the discrepancy in Ca2� uptake observed in the twomitochondrial populations.Ca2� uptake evaluated using both the infusion method and the bolus

additionswas dependent upon the electrogenicCa2� uniporter, as dem-onstrated by the addition of 600 nM RuRed, which totally preventedCa2� uptake (Fig. 8). In the presence of RuRed, therewas no loss of��mfollowing the addition of Ca2�.

Similar ROS Production between the Nonsynaptic and Synaptic Mito-chondria—Another mechanism that might underlie the different abilitiesof nonsynaptic and synaptic mitochondria to accumulate Ca2� is greaterROS production and oxidative damage in the synaptic population. Therewas no significant difference in basal ROS production, measured usingDCF, in isolated nonsynaptic and synaptic mitochondria (Fig. 9).

DISCUSSION

The results of this study demonstrate that nonsynaptic mitochondriaisolated from rat cortex can accumulate significantly more Ca2� than

FIGURE 4. Synaptic mitochondria are more susceptible to Ca2� overload than nonsynaptic mitochondria following a slow infusion of Ca2�. Isolated nonsynaptic or synapticmitochondria were placed in a constantly stirred, temperature-controlled cuvette inside a spectrofluorophotometer. CaG5N (A and C) and TMRE (B and D) were monitored simultaneously.Each sample was given malate and pyruvate as oxidative substrates, which caused an increase in ��m marked by a downward deflection (B). ADP caused a loss of some ��m as ADP isphosphorylated into ATP. Next oligomycin, the ATP synthase inhibitor, was added, and the mitochondria were at maximal ��m. Ca2� infusion began at 5 min (160 nmol of Ca2�/mg ofprotein) causing a small, initial increase in CaG5N fluorescence until the mitochondria were able to accumulate the added Ca2� (A and C). The nonsynaptic mitochondria (darker trace) wereable to buffer more of the infused Ca2� as compared with the synaptic mitochondria (lighter trace). By comparing the results obtained to the Ca2� signal observed in the absence of addedmitochondria (buffer, lightest trace), the modest Ca2� accumulation by the synaptic mitochondria is evident. Similar results were observed in isolated nonsynaptic and synapticmitochondria from SD (A and B) or F344 (C and D) rats. There was also a loss of ��m, as indicated by an increase in the TMRE fluorescence, earlier by the synaptic mitochondriafrom both strains, SD (C) and F344 (D). The y axis of the graphs is expressed in CaG5N or TMRE fluorescent arbitrary units (AU). These are representative traces from nonsynapticand synaptic mitochondria from the same preparation and this experiment was repeated in eight (SD) and five (F344) independent experiments.




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the synaptic mitochondria before undergoing mPT. This difference inCa2� accumulation was observed in two strains of rats utilizing bothbolus Ca2� additions and gradual Ca2� infusion paradigms. CsA, anmPT inhibitor, increased the amount of Ca2� that could be accumu-

lated in nonsynaptic mitochondria but did not have a significant effectin synaptic mitochondria. The uptake of Ca2� in both mitochondrialpopulations was dependent on the Ca2� uniporter and completelyinhibited by the addition of RuRed. The differences were not due to

FIGURE 5. Synaptic mitochondria were less able to buffer large bolus additions of Ca2� as compared with nonsynaptic mitochondria. Isolated nonsynaptic mitochondria(darker traces) from SD rats were able to accumulate more bolus additions of 1000 nmol Ca2�/mg (50 �M Ca2�) (A) and maintain ��m (B) longer than synaptic (lighter traces)mitochondria. When the mitochondria were no longer able to accumulate the added Ca2�, as evident by the increase in CaG5N signal (A) and the increase in TMRE (B), a bolus of EGTAwas added that increased ��m. Isolated synaptic mitochondria from F344 rats (C and D) demonstrated similar results. The y axis of the graphs is expressed in CaG5N or TMREfluorescent arbitrary units (AU). These data are representative traces from three independent experiments.




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initial Ca2� loads in the isolated mitochondria or the production ofmitochondrial ROS. There were no differences between nonsynapticand synaptic mitochondria in the rate of oxygen consumption, meas-ured during the classical states of respiration, indicating that the elec-tron transport chain was well coupled to oxidative phosphorylationallowing for the maintenance of normal ��m in both populations ofmitochondria.Previousmethods for enriching synapticmitochondria utilized either

Ficoll (19, 29, 40, 44) or Percoll (36) discontinuous density gradients anddetergents such as digitonin to disrupt synaptosomal membranes.Yields of synaptic mitochondria were low in the previous studies, pos-sibly because of the isolation protocol involving long centrifugationcycles (29) and damaging detergent techniques to release the synapticmitochondria from synaptosomes (18). In the present study, cortical ratbrain mitochondria were isolated from nonsynaptic and synapticsources using centrifugation on successive discontinuous Percoll gradi-ents and high pressure nitrogen cell disruption. The nonsynaptic andsynaptic mitochondria underwent similar manipulations during theirenrichment; both populations were exposed to the nitrogen disruption,and both underwent centrifugation through two discontinuous Percollgradients. Use of the nitrogen disruption method (37) avoided the del-eterious effects of detergents on mitochondrial proteins (18).Synaptic mitochondria are largely presynaptic, because the homoge-

nization procedure pinches off presynaptic terminals at the neck of the

axon, and postsynaptic mitochondria are not retained in the synapto-somes (47, 48). The mitochondria within the synaptic fraction arederived from both interneurons and projection neurons from subcorti-cal regions. Within the presynaptic bouton, mitochondria serve a num-ber of functions. In addition to ATP synthesis and Ca2� buffering, theycontribute to neurotransmitter synthesis and catabolism. The bioener-getic demands in the presynaptic terminal are high, with ATP beingrequired for endo- and exocytosis, in addition to maintenance of ionhomeostasis. Presynaptic mitochondria are constantly exposed to highCa2� transients associated with transmitter release (49).

Nonsynaptic mitochondria originate from both neurons and non-neuronal cells. In rat parietal cortex, glial cell density is approximatelydouble the neuronal density, and mitochondria occupy a smaller per-centage of the cytosol in glia as compared with neurons (50). Using asimilar method to that used in the present study to isolate nonsynapticmitochondria, Kristian and colleagues (51) found faint peripheral ben-zodiazepine receptor immunoreactivity, a marker of astrocytic mito-chondria (52), in the nonsynaptic mitochondrial fraction in contrast tostrong immunoreactivity in mitochondria isolated from cultured astro-cytes. The weak peripheral benzodiazepine receptor immunoreactivityis not due to decreased expression in adult rats (52), indicating that thenonsynaptic mitochondrial fraction may be predominantly neuronal.At present, it is not possible to isolate mitochondria from neuronsversus glia in the adult CNS. Although mitochondria can be isolated

FIGURE 6. Comparison of mitochondrial Ca2� accumulation following infusion and bolus Ca2� addition in synaptic versus nonsynaptic mitochondria. Representative Ca2�

infusion traces from F344 rat cortical mitochondria, in the presence and absence of CsA, are shown in A. The y axis of the graph is expressed in CaG5N fluorescence arbitrary units (AU).Similar results were obtained in SD rats (not shown). In B–D, the y axis indicates the amount of Ca2� added to the mitochondrial suspension. Using the 50% above base-line thresholdcalculation described under “Materials and Methods” and illustrated in A to quantify the amount of Ca2� added prior to the mitochondria undergoing permeability transition, the SDand F344 infusion data were analyzed by using an unpaired t test (B). There were no significant differences between the nonsynaptic SD (n 10) and the nonsynaptic F344 (n 5)or between the synaptic SD (n 8) and the synaptic F344 (n 5). Therefore all of the traces from the two strains were combined for subsequent analysis (C). The data from all of thestrains were analyzed using a one-way analysis of variance with a Scheffe’s post-hoc analysis. The nonsynaptic mitochondria (n 15) were exposed to more Ca2� than the synapticmitochondria (n 13) prior to undergoing mPT (p � 0.0001). The addition of CsA enabled nonsynaptic mitochondria (n 6) to withstand greater accumulated more Ca2� than thenonsynaptic alone (p � 0.0036) or the synaptic alone (p � 0.0001) or the synaptic with CsA (n 5) (p � 0.0001). The synaptic mitochondria with CsA were not significantly differentfrom the synaptic mitochondria alone. D, finally, nonsynaptic (n 5) and synaptic (n 5) mitochondrial traces using the bolus addition paradigm were quantitated using an unpairedt test. The nonsynaptic mitochondria accumulated significantly more bolus additions of Ca2� than the synaptic mitochondria. Each group within all of these analyses was representedby at least five independent experiments.




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from primary cultures of neurons and astrocytes (51), the yields are low,and the Ca2� handling abilities of the two mitochondrial populationshave not been evaluated.The differences in mitochondrial Ca2� handling were evident fol-

lowing both bolus Ca2� additions and continuous Ca2� infusion tothe isolated synaptic and nonsynaptic mitochondria.With bothmeth-ods, a loss of��mpreceded or accompanied the impairment inmitochon-drialCa2� accumulation, similar to results previously obtained in brain andliver mitochondria (5). Using the bolus additions paradigm, there arehigh bioenergetic consequences because of immediate changes on theproton gradient (5). In addition, the various calcium phosphate salts(mono-, di-, and tri-calcium orthophosphate and hydroxyapatite) pro-duced in response to the bolus addition may differ from those resultingfrom a slow accumulation of Ca2� (5). Use of a gradual infusion tech-nique allows formitochondria to slowly accumulate the Ca2� withmin-imal bioenergetic consequences and thereby allows a more accurateestimate of the Ca2� buffering capacity of the mitochondria (5, 53).However, evenwith slow infusion, the amounts of Ca2� added aremuchgreater than physiological free Ca2� cytosolic levels in presynaptic ter-minals, which are �100 nM at rest and estimated to reach 8.5 �M fol-lowing sustained depolarization in the mouse calyx of Held, a largeglutamatergic CNS terminal (54). The mechanism underlying thegreater amount of Ca2� accumulation following bolus Ca2� addition ascompared with gradual infusion is uncertain. Differences in Ca2�

uptake may reflect increased recovery times in experiments using thebolus additions, whereasmitochondria are continuously exposed to ele-vated Ca2� in the infusion paradigm. It is interesting to note that theuptake dynamics following the first bolus addition was identical betweenthe two populations, but that uptake was progressively decreased in thesynaptic population with successive bolus Ca2� additions, suggesting thata subpopulationof the synapticmitochondriaundergoespermeability tran-sition with each Ca2� bolus. This is currently being investigated in ourlaboratories. Alternatively, formation of various forms of Ca2� phos-

phate (5, 55) could contribute to differences in Ca2� accumulation abil-ity between the nonsynaptic and synaptic mitochondria in the twoparadigms.The difference in Ca2� handling between isolated synaptic and

nonsynaptic CNS mitochondria has not been examined previously,although there is substantial evidence for differences in Ca2� buff-ering and mPT induction among various neural and non-neuralmitochondrial populations. Isolated brain mitochondria are moreresistant to mPT induction as compared with liver mitochondria (14,15, 56–58). However, someof this resistancehasbeenattributed to theuseof strong detergents in the preparations and may not reflect differences inthe mitochondria themselves (45). Across brain regions, variability toundergo Ca2�-induced dysfunction has been observed (16–18). This maybe due, at least in part, to differing levels of cyclophilin D, a peptidyl-prolylcis-trans isomerase that facilitates Ca2�-induced permeabilization of theinner mitochondrial membrane and mPT pore formation (59, 60). CsAantagonizes mPT by binding to cylophilin D (61, 62). We previouslyobserved a difference in the response to CsA in isolated nonsynapticcortical versus spinal cord mitochondria consistent with increasedcyclophilin D mRNA expression in spinal cord (43). The cyclophilin Dcontent of synaptic and nonsynaptic mitochondria remains to be deter-mined. However, the response of the two mitochondrial populations toCsA suggests that alternate or additional mechanisms are likely respon-sible for the difference in Ca2� handling. Oxidation of the adenosinenucleotide translocator, a major component of the mPT pore, can alsofacilitate mPT (43, 63). Mitochondria in the presynaptic terminal mustbe transported from the cell body. As they age, mitochondria undergooxidative damage and become depolarized (27). Mitochondria with ahigh ��m are transported toward the presynaptic terminal via fastaxonal transport, whereas mitochondria with low potential are trans-ported retrogradely to the soma to be degraded (64). Thus, a possibleexplanation for the greater propensity of synaptic mitochodria toundergo Ca2�-induced mPT is their increased age and oxidation,although this remains to be determined experimentally.The impaired Ca2� uptake capacity of synaptic mitochondria may

also reflect their function andmorphology. The extent ofCa2� bufferingby neuronal, presynapticmitochondria has been a subject of debate (65).Synapticmitochondriamay dampen large elevations in Ca2� associatedwith repetitive synaptic activity (66–70). However, there is also evi-dence that Ca2� release from mitochondria can potentiate transmitterrelease (71). Within the large calyx of Held brainstem terminal, mito-chondria are associated with a specialized adherens complex implicatedin exocytosis and endocytosis (72). Similar structures are evident inother CNS presynaptic terminals (73). Presynaptic mitochondria aretypically punctuate and isolated, in contrast to the mitochondrialthreads found in neuronal dendrites and the mitochondrial clustersobserved in neuronal soma (21, 22). Within primary cortical astrocytes,mitochondria are predominantly observed as threads, although isolatedmitochondria can be found in the periphery (22). Dynamin-related pro-tein 1 is required for mitochondrial fission (74, 75) but also reducesmitochondrial Ca2� retention capacity (76). In the Drosophila drp1mutant, the loss of mitochondrial fission results in a near absence ofsynaptic mitochondria (77). This does not influence basal synapticproperties but impairs the ability to mobilize reserve pool vesicles, andthis is partially rescued by the addition of exogenousATP. Together, theabove results suggest that the primary function of synaptic mitochon-dria to maintain high ratios of ATP:ADP that can be utilized for exocy-tosis and endocytosis. The limited ability of synaptic mitochondria tobuffer large Ca2� loads may result from their punctuate morphology, in

FIGURE 7. Addition of the uncoupler CCCP demonstrates similar Ca2� levels withinthe mitochondrial matrix of synaptic and nonsynaptic mitochondria. CCCP wasadded at 7 min to cause release of Ca2� into the buffer, detected as an increase in CaG5Nfluorescence (A) and complete loss of the ��m, assessed using TMRE (B). These repre-sentative traces are from the same mitochondrial preparation, and this experiment wasrepeated in two independent experiments.




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contrast to the tubular mitochondrial morphology evident in otherregions of neurons and glia.The reduced Ca2� buffering capacity of synaptic mitochondria and

their increased propensity to undergo mPT may also have pathologicalconsequences. Degeneration of distal axon segments is one of the initialevents observed in several neurodegenerativedisorders including amyotro-phic lateral sclerosis, spinocerebellar disorders, intoxications, and AIDS(78). Moreover, synapse elimination occurs early in the progression ofAlzheimer disease (79, 80) and is also implicated in schizophrenia (81).

It is clear that synapse and neurite degeneration can occur independ-ently of cell death (82), and although themechanisms are unclear, mito-chondria are strongly implicated (83). In cultured hippocampal neuronsexposed to glutamate, synapse loss precedes cell death (84). An age- ordisease-related decline in mitochondrial Ca2� buffering capacity (17,85) may further increase the vulnerability of presynaptic terminals tomPT and subsequent release of death-related proteins (2, 80, 86) andthereby contribute to the pathogenesis of late onset neurodegenerativedisorders.

FIGURE 8. Addition of uniporter inhibitor RuRedblocks Ca2� uptake of Ca2� in both nonsynapticand synaptic mitochondria. Using the sameexperimental paradigm described in the legendto Fig. 4, 600 nM RuRed was added at the begin-ning of the trace (A) or at 5 min (B) to inhibit theCa2� uniporter. This blocked uptake of Ca2� intoboth nonsynaptic and synaptic mitochondriareceiving Ca2� through either the infusion pump(A) or bolus additions (B). The y axis of the graphs isexpressed in CaG5N or TMRE fluorescent arbitraryunits (AU). These are representative traces fromthe same mitochondrial preparation, and thisexperiment was repeated in two independentexperiments.




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In summary, the results of the present study demonstrate that mito-chondria isolated from rat cortical synaptosomes have an increased pro-pensity to undergo mPT in response to added Ca2� as compared withmitochondria isolated from a nonsynaptosomal fraction. This differ-ence is not the result of differences in mitochondrial bioenergetics orinitial Ca2� load but may reflect the largely neuronal origin of synapticmitochondria versus themixed cellular origin of nonsynapticmitochon-dria or the different functions of synaptic versus nonsynapticmitochon-dria. It is also possible that the isolated nature of synaptic mitochondriain comparison with the threads and clusters found in other cellularlocations, alterations in cyclophilin D content, or the greater age andcumulative oxidative damage to synaptic mitochondria may underliethe differences in Ca2� accumulation. Although the precise mecha-nisms underlying the Ca2� handling differences remain to be deter-mined, the results provide an explanation for the vulnerability of distalaxon segments and presynaptic terminals in several neurodegenerativedisorders and following neuronal insult.

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Maile R. Brown, Patrick G. Sullivan and James W. GeddesMitochondria

Overload than Nonsynaptic2+Synaptic Mitochondria Are More Susceptible to Ca

doi: 10.1074/jbc.M510303200 originally published online March 3, 20062006, 281:11658-11668.J. Biol. Chem.

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