Mutant Huntingtin Expression Induces Mitochondrial Calcium

Mutant Huntingtin Expression Induces MitochondrialCalcium Handling Defects in Clonal Striatal CellsFUNCTIONAL CONSEQUENCES*

Received for publication, April 21, 2006, and in revised form, August 2, 2006 Published, JBC Papers in Press, September 13, 2006, DOI 10.1074/jbc.M603845200

Tamara Milakovic, Rodrigo A. Quintanilla, and Gail V. W. Johnson1

From the Department of Psychiatry, University of Alabama at Birmingham, Birmingham, Alabama 35294

Huntington disease (HD) is caused by a pathological elonga-tion of CAG repeats in the huntingtin protein gene and is char-acterizedby atrophy andneuronal loss primarily in the striatum.Mitochondrial dysfunction and impaired Ca2� homeostasis inHD have been suggested previously. Here, we elucidate the effectsof Ca2� on mitochondria from the wild type (STHdhQ7/Q7) andmutant (STHdhQ111/Q111) huntingtin-expressing cells of striatalorigin. When treated with increasing Ca2� concentrations,mitochondria frommutant huntingtin-expressing cells showedenhanced sensitivity to Ca2�, as they were more sensitive toCa2�-induced decreases in state 3 respiration and ��m, thanmitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2� uptakecapacity in comparison with wild type cells. Decreases in state 3respirationwere associatedwith increasedmitochondrialmem-brane permeability. The ��m defect was attenuated in thepresence ofADPand thedecreases inCa2�uptake capacitywereabolished in the presence of Permeability TransitionPore (PTP)inhibitors. These findings clearly indicate that mutant hun-tingtin-expressing cells have mitochondrial Ca2� handlingdefects that result in respiratory deficits and that theincreased sensitivity to Ca2� induced mitochondrial perme-abilization maybe a contributing mechanism to the mito-chondrial dysfunction in HD.

Huntington disease (HD)2 is a neurodegenerative diseasethat is inherited in an autosomal dominant manner. Itbelongs to a family of CAG expansion diseases and is causedby the pathological elongation of the CAG repeats in exonone of the huntingtin protein gene (1). Symptoms and dis-ease progression are caused by dysfunction and loss of neu-

rons starting in the striatum (specifically medium spiny neu-rons), but progressing to cortex and to a lesser extent toother brain regions in the later stages of the disease (2). Dis-ease is caused by the toxic gain of function of mutant proteinbut some loss of function may also contribute to the patho-genesis (for review see Ref. 3). The toxic gain of function ofmutant huntingtin has not been clearly defined, but there arefindings suggesting that mutant huntingtin causes transcrip-tional dysregulation (4), ubiquitin-proteasome system dys-function (5), Ca2� homeostasis dysfunction (6, 7), and mito-chondrial dysfunction (7–10).Mitochondrial dysfunction in HD has been suggested

primarily by the studies showing impairment of mitochondrialcomplexes (II, III, and IV) specifically in the striatum in the latestages HD patients (8–10). Administration of the mitochon-drial complex II inhibitor 3- nitropropionic (3-NP) in bothrodents and nonhuman primates resulted in symptoms andneuropathology that resemble HD (11, 12). Further, studieshave shown impairment of mitochondrial Ca2� buffering inHD lymphoblast cell line and brainmitochondria from the full-length mutant huntingtin transgenic mice (YAC72) (7, 13).Striatum, the primary region to get affected in HD is highly

innervated by cortical glutaminergic projections (2). Previouslyit has been demonstrated that mitochondrial dysfunction canlead to neuronal sensitization to glutamate leading to excito-toxic cellular dysfunction and cell death (14, 15). Hence, eventhough HD is not a classic mitochondrial disease (16), elucida-tion of mitochondrial dysfunction mechanisms would likelyprovide important insight in HD pathogenesis.To study the effects of mutant huntingtin on mitochondrial

function, conditionally immortalized cells of striatal origin thatexpress endogenous, comparable levels of either wild type(STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin wereused (17). These cell lines are prepared fromwild type (HdhQ7/Q7)and mutant huntingtin knock-in mice (HdhQ111/Q111) (17) andtherefore the STHdhQ111/Q111 cell line is a genetically accuratecellmodel ofHD. In our previous study (18), we investigated theeffects of mutant huntingtin on mitochondrial electron trans-port chain complexes using STHdhQ7/Q7 and STHdhQ111/Q111

cell lines. Given the fact that the metabolic thresholds andenzyme activities of electron transport chain complexes werenot different between the two cell lines, it is likely that themito-chondrial complex deficits are a later event in the course of HDpathogenesis, indeed in low grade HD cases no deficits in theenzyme activities of electron transport chain complexes wereobserved (19).

* This study was supported by National Institutes of Health Grant NS041744.The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

1 To whom correspondence should be addressed: Dept. of Psychiatry,1720 7th Ave. South, SC 1061, University of Alabama at Birmingham,Birmingham, AL 35294-0017. Tel.: 205-934-2465; Fax: 205-934-3709;E-mail: [email protected].

2 The abbreviations used are: HD, Huntington disease; ��m, mitochondrialmembrane potential; PTP, permeability transition pore; 3-NP, 3-nitropro-pionic acid; KRH, Krebs-Ringer-HEPES; CM-H2TMRos, MitoTracker® Red;JC-1,5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazolylcarbocyanineiodide; RCR, respiratory control ratio; FCCP, carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone; ROS, reactive oxygen species; 4-BrA23187,4-bromo A-23187, free acid; ANT, adenine nucleotide translocator; ANOVA,analysis of variance; Cab, Ca2� uptake buffer.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 34785–34795, November 17, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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In the present study, we examined the effects of Ca2� onmitochondria from STHdhQ7/Q7 and STHdhQ111/Q111 cells.Isolated mitochondria were treated with increasing Ca2� con-centrations andmitochondrial function was assessed using dif-ferent assays. We determined that mutant huntingtin-express-ing cells have decreased Ca2� uptake capacity, and exhibitincreased sensitivity to Ca2�-induced decreases in respirationand ��m. The ��m defect was attenuated in the presence ofADP and the decrease in Ca2� uptake capacity was abolished inthe presence of Permeability Transition Pore (PTP) inhibitors.This study clearly demonstrates that mitochondrial Ca2� buff-ering capacity in STHdhQ111/Q111 cells is compromised, andsuggests increased sensitivity to Ca2�-induced mitochondrialpermeabilization as amechanismofmitochondrial dysfunctionin HD.

EXPERIMENTAL PROCEDURES

Materials—All chemicals were from Sigma-Aldrich unlessotherwise noted. All buffers used in experiments with crudemitochondrial preparations were prepared in water (Sigma,catalogue no. 95305) that is standardized for Ca2� content([Ca2�]� 0.000001%).Cell Culture—In this study, conditionally immortalized

striatal progenitor cell lines: STHdhQ7/Q7 cell line expressingendogenous wild type huntingtin and the homozygousmutant STHdhQ111/Q111 cell line expressing comparable lev-els of mutant huntingtin with 111 glutamines were used. Celllines were prepared from wild type mice and homozygousHdhQ111/Q111 knock-in mice and were described previously(17). Culturing conditions were the same as described in ourprevious study (18).Isolation of Mitochondria—Cells were grown on 150-mm

plates until � 80–90% confluency, washed twice with cavita-tion buffer (250 mM sucrose, 5 mM HEPES, 3 mM MgCl2, 1 mMEGTA, pH 7.3 corrected with 5 M KOH) and scraped into cav-itation buffer using soft rubber scrapers. Cells were openedusingN2 cavitation for 5min at 250 psi on ice, and sampleswereadditionally homogenized with 1 stroke in a glass Douncehomogenizer. Homogenates were centrifuged at 7000 � g for10 min at 4 °C. Supernatants were aspirated, and pellets wereresuspended in cavitation buffer and used as crude mitochon-drial preparations. Protein concentrations in crude mitochon-drial preparations were determined using the bicinchoninicacid assay (Pierce), and aliquots were then prepared that con-tained the indicated protein content for each measurement.Aliquots were centrifuged at 7000 � g for 10 min and kept onice in cavitation buffer until use in each assay.Measurement of Mitochondrial Respiration—Respiration

rates were measured using an oxygraph (Hansatech Instru-ments) as described previously (18). Crudemitochondrial prep-arations (0.5-mg aliquots) were resuspended in respirationbuffer (130 mM KCl, 20 mMHEPES, 2 mMMgCl2, 2 mM EGTA,2 mM potassium phosphate (KH2PO4/K2HPO4, 1:1.78), 1%essentially fatty acids free bovine serum albumin, pH 7.2adjusted with 5 M KOH) to a final concentration of 1 mg/ml.The mitochondrial suspension (0.5 ml volume) was placed inthe respiratory chamber and allowed to equilibrate for 2 min.Respiratory substrate (glutamate (10 mM) plus malate (10 mM)

or succinate (5mM)with rotenone (10�M)) was then added andstate 4 respiration was measured for 2 min, ADP (1.5 mM) wasthen added, and state 3 respiration was measured for a further2–4 min. Rates were normalized to citrate synthase activity inthe same samples. Citrate synthase activity was determined aspreviously described (18).Ca2�Titration Experiments—Respiration buffers containing

specific free Ca2� concentrations (Ca2�-EGTA respirationbuffers) were prepared on the day of the experiment. To calcu-late the amount of total Ca2� that was needed to achieve theappropriate free Ca2� concentration in the respiration bufferthat contained 2 mM EGTA we used MaxChelator software(20). Each Ca2�-EGTA respiration buffer was prepared sep-arately by diluting each specific 100� CaCl2 stock in therespiration buffer and correcting its pH to 7.2 using 0.1 MKOH in the respiration buffer. CaCl2 stocks were preparedfrom CaCl2�2H2O (minimum 99%), that was dried overnightand stored in a desiccation chamber until use. Crudemitochon-drial preparations were dissolved in prepared Ca2�-EGTA res-piration buffers, and respiration rates were measured asdescribed above. The period between buffer addition to themitochondrial preparation and initiation of state 3was�5min.

Free Ca2� concentrations in the Ca2�-EGTA buffers werechecked using a calibrated Ca2� electrode on the day of theexperiment. Measured concentrations were averaged and pre-sented on the X-axis of Ca2� titration experiments graphs.Actually concentrations were always slightly higher than thosecalculated by software.Cytochrome c and NADH Respiration Experiments—Respi-

ration experiments were performed as described above. State 3respiration was measured for 2 min prior to the addition ofcytochrome c (30 �M), and respiration was monitored foranother 2 min. This was followed by the addition of NADH (5mM), and respiration was monitored for an additional 2 min.Determination of Mitochondrial Ca2� Uptake Capacity—

Ca2� uptake capacities were measured using a Ca2� electrode(World Precision Instruments). Crude mitochondrial prepara-tion was resuspended in Ca2� uptake buffer (130 mM KCl, 20mM HEPES, 2 mM MgCl2, 2 mM potassium phosphate(KH2PO4/K2HPO4, 1:1.78), 1% bovine serum albumin, pH 7.2adjusted with 5 M KOH) and placed in the oxygraph respiratorychamber. The respiratory chamber was thermostatted at 37 °C,and its contents were constantly mixed with an electromag-netic stirrer bar. Glutamate (10 mM) and malate (10 mM) wereadded as respiratory substrates. Ca2� and reference electrodeswere added to the chamber from the top. Starting volume of thereactionwas 2ml. The chamberwas kept open during an exper-iment. Ca2� additions were performed using fine tubing and aHamilton syringe. 5, 10, and 20 mM CaCl2 stocks were used tomake 10, 20, 40, or 80 nmol of Ca2� additions. The Ca2� elec-trode measures extramitochondrial Ca2� and increases in thesignal present as downward deflections on the traces.To observe the effects of PTP inhibition on Ca2� uptake

capacity we used cyclosporine A (1 �M) plus ADP (50 �M) plusoligomycin (2 �g/ml). The PTP inhibitors were added to therespiratory chamber prior to Ca2� additions (21).

To calculate Ca2� uptake capacity, we counted number ofCa2� additions until the addition after which no uptake was

Ca2� Handling Defects with Mutant Huntingtin Expression

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observed (trace horizontal). The number of additions was mul-tiplied by the nmol of Ca2� per addition, and normalized toprotein content.Mitochondrial Membrane Potential (��m) Determination

in Live Cells—Mitochondrial membrane potential was esti-mated using the specific mitochondrial probes: MitotrackerRed (CM-H2TMRos) and tetramethylrhodamine ethyl ester(TMRE) (Molecular Probes) (22–26). Cells were grown onpoly-L-lysine-coated plates and cultured for 3 days. The cellswere then loaded for 30 min with CM-H2TMRos in KRH-glu-cose, washed, and allowed to equilibrate for 15 min. Cell plateswere thenmounted in a chamber on the stage of a confocal laserscanning microscope (Leica model TCS SP2). Quantitativemeasurements of CM-H2TMRos fluorescence were performedby confocal microscopy (Leica model TCS SP2), using a 40�water immersion lens. CM-H2TMRos fluorescence imageswere obtained by excitation at 563 nm, reflection off a dichroicmirror with a cut-off wavelength at 564 nm, and longpass emis-sion filtering at 590 nm. For TMRE experiments, cells wereloaded with TMRE (100 nM) for 45 min in KRH-glucose, andthen were mounted on the stage for confocal microscopy.TMRE fluorescencewas detected excitingwith a 561nmHe-Nelaser line very heavily attenuated (10% laser power), and theemission was collected at �563 nm (22). Signal from controlcells and cells treated with different stimuli were comparedusing identical settings for laser power, confocal thickness, anddetector sensitivity for each dye and separate experiment (23,25, 26). The images were analyzed with LCS Leica confocalsoftware and recorded as the mean Mitotracker Red or TMREfluorescence signal per live cell.Measurement of Mitochondrial Membrane Potential

(��m) inMitochondrial Preparations—��mwas measuredusing 5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimida-zolylcarbocyanine iodide (JC-1; Molecular Probes) accord-ing to a published protocol with modifications (27). Modifi-cations were made so that method could be used withisolated mitochondria. To measure ��m at different Ca2�

concentrations, crude mitochondrial preparation was ali-quoted into the wells of 96-well plate (50 �g/well). The platewas centrifuged at 3220 � g for 10 min at 4 °C, and superna-tants were carefully aspirated. Ca-EGTA respiration bufferswith 0, 0.4, or 0.6 �M (software calculated) free Ca2� or Ca2�

uptake buffer with 150, 500, or 1000 �M Ca2� each supple-mented with glutamate (10 mM), malate (10 mM), and with(for state 3) or without (for state 4) ADP (1.5 mM) were addedto separate wells in duplicates (50 �l/well) and incubated at37 °C for 10 min. Supernatants were carefully aspirated, andthe same buffers but containing JC-1 (5 �g/ml) were addedto the wells (50 �l/well). Wells in which FCCP (20 �M) wasalso added were considered as positive controls. The platewas incubated for 30min, at 37 °C in dark, supernatants wereaspirated, and fluorescence was read at 485/528 nm and 530/590 nm. The ratio between the fluorescence was used todescribe ��m as published previously (27).Measurement of Mitochondrial H2O2 Production—To deter-

mine mitochondrial ROS production we used an Amplex Red(Molecular Probes) assay (28). Crude mitochondrial prepara-tions were aliquoted (100 �g/well) and pelleted onto a 96-well

plate as described for measuring ��m. Mitochondrial pelletswere covered with Ca-EGTA respiration buffers with 0, 0.4, or0.6 �M (software calculated) free Ca2� or Ca2� uptake bufferwith 150, 500, or 1000 �MCa2� each supplemented with gluta-mate (10 mM), malate (10 mM), Amplex Red (50 �M), horserad-ish peroxidase (0.01 units/ml or 0.1 units/ml), and with (forstate 3) or without (for state 4) ADP (1.5 mM). Plate was read inthe kineticmode for 30min at excitation/emissionwavelengths530/590 nm at 37 °C. Rates of H2O2 production were deter-mined using a standard curve.Statistical Analysis—Results were analyzed using ANOVA,

Student’s t test, or paired t test as indicated. Differences wereconsidered significant if p � 0.05.

RESULTS

Effects of Ca2� on Respiration in Mitochondria from STH-dhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant) Cells—Ithas been shown previously that at the free concentrationshigher than 1 �M, Ca2� causes strong inhibition of oxidativephosphorylation (29). To determine the effects of Ca2� on oxi-dative phosphorylation in mitochondria isolated from the cellsexpressing endogenous levels of wild type (STHdhQ7/Q7) ormutant (STHdhQ111/Q111) huntingtin, wemeasured state 4 andstate 3 respiration rates in respiration buffers containingincreasing free �M Ca2� concentrations. In these experiments,EGTA-based respiration buffer was used for 0 �M Ca2� andCa2�-EGTA respiration buffers were prepared as describedunder “Experimental Procedures.” At 0 �M Ca2�, we observedno differences in the state 4 or state 3 respiration rates betweenwild type and mutant cells when glutamate plus malate (com-plex I substrate) or succinate (complex II substrate) were usedas substrates (Fig. 1A). As described earlier (29), with increasingfree �MCa2� concentrations decreases in the state 3 rates wereobserved (Fig. 1B). However, this decrease was more pro-nounced in the mitochondria from the mutant huntingtin-ex-pressing cells, reaching significance at lower Ca2� concentra-tions than in the wild type (Fig. 1B). State 4 rates increased withincreasing Ca2� concentrations, reaching significance only inthemutant at the highest Ca2� concentration used (Fig. 1B). Todescribe overall changes in the respiration rates, we calculatedRespiratory Control Ratios (RCRs) at the different Ca2� con-centrations. RCRwas calculated as the ratio between state 3 andstate 4 rates. A decrease in RCR was observed with increasingCa2� concentrations and was more pronounced in mitochon-dria frommutant cells, reaching significance at the lower Ca2�

concentrations than in the wild type cells (Fig. 1C). Theseresults indicate that mitochondria from STHdhQ111/Q111

(mutant) cells are more sensitive to Ca2�-induced changes inoxidative phosphorylation than mitochondria from STH-dhQ7/Q7 (wild type) cells.Ca2� Uptake Capacity in Mitochondria from STHdhQ7/Q7

(Wild Type) and STHdhQ111/Q111 (Mutant) Cells—Severalstudies have suggested that there is reduced mitochondrialCa2� buffering capacity in HD. Panov et al. (7, 13) demon-strated diminished Ca2� uptake capacity inmitochondria fromHD lymphoblast cell lines, and brain mitochondria from thefull-length mutant huntingtin-overexpressing mice (YAC72)(7), whereas others demonstrated diminished Ca2� uptake in



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muscle mitochondria from R6/2 mice (30). To comprehen-sively describe the effects of Ca2� on mitochondria in ourmodel, we determined mitochondrial Ca2� uptake capacity inSTHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells.For these experiments we used a Ca2�-sensitive electrode, asdescribed under “Experimental Procedures.” To determine,mitochondrial Ca2� uptake, isolated mitochondria (1.5 mg/2ml) were placed in a 37 °C thermostatted chamber and chal-lenged with 10-nmol Ca2� pulses every 3 min. Representativetraces are shown in Fig. 2C. Ca2� uptake capacity was calcu-lated as described under “Experimental Procedures.” Weobserved that mitochondria from STHdhQ111/Q111 (mutant)cells have significantly diminished Ca2� uptake capacity com-pared with mitochondria from STHdhQ7/Q7 (wild type) cells

(Fig. 2A). To determine the “initial uptake” rates we calculatedthe average of the rates after the second, third, and fourth addi-tions of Ca2� and determined that the “initial uptake” rateswere significantly diminished in the mitochondria from themutant cells (Fig. 2B). These results indicate that mitochondriafrom STHdhQ111/Q111 (mutant) cells have a Ca2�-bufferingdefect, as they can take up less Ca2� than the mitochondriafrom wild type cells. Because ER contamination of the mito-chondrial preparationwas a possibility, we confirmed themito-chondrial nature of the Ca2� uptake in our mitochondrialpreparations, as the addition of uncoupler (FCCP) causedrelease of Ca2�, and pretreatment of the cells with thapsigargin(which blocks the Ca2� uptake pump of the ER Ref. 31) did notproduce any change in the Ca2� uptake capacity (Fig. 2D).

FIGURE 1. Effects of Ca2� on respiration in mitochondria from STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, state 4 (st4) and state 3 (st3)respiration rates measured in crude mitochondrial preparations in EGTA-based respiration buffer. Crude mitochondria from wild type and mutant cells wereincubated with either glutamate plus malate (glu.�mal.) or succinate with rotenone (succ.�rot.) as respiratory substrates, and respiration rates were deter-mined as described under “Experimental Procedures.” Rates were normalized to citrate synthase activity measured in the same samples. No significantdifferences were observed. B, respiration rates measured in the presence of increasing free Ca2� concentrations. Crude mitochondria were resuspended inCa-EGTA buffers with the indicated free Ca2� concentrations, and state 4 and state 3 rates were determined as described under “Experimental Procedures.”Glutamate plus malate was used as the respiratory substrate. Results are expressed as percentage of the state 3 rates at 0 Ca2� for each of the cell lines.Significant decreases in state 3 rates at the low �M free Ca2� concentrations were observed, with the decreases occurring at the lower Ca2� concentrations inmitochondria from the mutant cells compared with wild type mitochondria. State 4 rates showed a trend toward increasing with increasing free Ca2�

concentrations, with significance being reached only at the highest Ca2� concentration used in the mutant cells. C, RCRs in the presence of different free Ca2�

concentrations were calculated as ratios between state 3 and state 4 rates presented in B. The decrease in RCR reached significance at lower Ca2� concentra-tions in mutant cells than in the wild type group. All data are mean S.E. of 3– 4 independent experiments. For statistical analyses ANOVA followed by theTukey post test (*, p 0.05) was used.



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Analysis of Mitochondrial Membrane Integrity Before andAfter Ca2� Addition in STHdhQ7/Q7 (Wild Type) and STH-dhQ111/Q111 (Mutant) Cells—Ca2� overload of mitochondriaresults in increased mitochondrial membrane permeability(32). To further study the cause of differences between mito-chondria from wild type and mutant cells in their sensitivityto Ca2�, we wanted to determine if the decrease in respira-tion observed in the presence of free �M Ca2� concentra-tions was associated with increased permeability of themito-chondrial membrane. First, we analyzed the integrity ofmitochondrial membrane in the basal conditions. NADH isthe substrate for mitochondrial complex I. However, theinner mitochondrial membrane is not permeable to exogenousNADH (33). When pyruvate plus malate was added to providereduced adenine dinucleotides (NADH, FADH2) inside themitochondria we observed significant state 3 rates upon ADP

addition (Fig. 3A). However, when NADH was used as the res-piratory substrate, we did not observe induction of state 3 res-piration in mitochondria from any of the cell lines (Fig. 3A).Representative traces are shown (Fig. 3A). This indicates goodintegrity of mitochondrial inner membrane in basal conditionsin mitochondria from both cell lines.It has been suggested that damage of the outer mitochon-

drial membrane results in the activation of alternative respi-ratory pathway in the presence of exogenous NADH andcytochrome c (34–37). In this pathway, NADH is oxidized atthe outer mitochondrial membrane leading to reduction ofexogenous cytochrome c. As described previously, if outermembrane is being compromised, cytochrome c will translo-cate to the complex IV and stimulate respiration (35). To assessmitochondrial membrane integrity, we measured the effects ofNADH and cytochrome c on state 3 respiration in the absence

FIGURE 2. Ca2� uptake capacity measured in mitochondria from STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, Ca2� uptake capacities.Ca2� uptake was measured using a Ca2�-sensitive electrode as described under “Experimental Procedures.” Crude mitochondria (1.5 mg in 2 ml) were given10-nmol Ca2� pulses every 3 min, until uptake could not be observed anymore. Ca2� uptake capacities were calculated by multiplying the number of pulsesby 10 nmol and normalizing it to mg of protein. Significantly lower Ca2� uptake capacity in mitochondria from mutant cells as compared with mitochondriafrom wild type cells was observed. B, initial Ca2� uptake rates. Initial uptake rates were calculated by averaging rates of uptake after the second, third and fourthadditions of Ca2�. Initial Ca2� uptake rates were significantly lower in mitochondria from mutant cells compared with wild type cells. Data are mean S.E. ofthree independent experiments. Student’s t test (*, p 0.05) was used for statistical analyses. C, representative traces of Ca2� electrode recordings (Ca2�

uptake is reflected by an upward deflection of the trace). Small arrows indicate Ca2� additions. Large arrows indicate the last pulses included in the calculations.D, control experiment that demonstrate the mitochondrial nature of Ca2� uptake measured by Ca2�-sensitive electrode in the crude mitochondrial prepara-tion. Mitochondria from mutant cells were used in these control experiments. Addition of FCCP caused release of Ca2� from mitochondria as evident byincrease in [Ca2�] in the reaction buffer (downward deflection of the trace after FCCP). Thapsigargin (1 �M) pretreatment of the cells for 30 min did not affectCa2� uptake capacity.



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or presence of Ca2�. For these experiments, we chose Ca2�

concentration that caused a significant decrease in state 3 ratein the mitochondria from both cell lines. Experiments werecarried out as described under “Experimental Procedures.” Inthe absence of Ca2�, neither cytochrome c alone nor cyto-chrome c plus NADH had any effect on respiration (Fig. 3B).This indicated good integrity of the outer mitochondrial mem-brane in both cell lines. However in the presence of Ca2�, when

the state 3 respiration was de-creased, cytochrome c plus NADHcaused a significant increase in state3 (Fig. 3B). Alamethicin, formspores in the membrane, was used asa positive control for the method(not shown).3 Cytochrome c alonedid not affect state 3 respiration inthe presence of Ca2� (Fig. 3B), sug-gesting that no substantial lossof cytochrome c is causing thedecrease in state 3 rate. Theseresults suggest that mitochondriafrom both wild type and mutantcells do have goodmembrane integ-rity in the absence of Ca2�. Further,the decrease in state 3 rates in thepresence of �M Ca2� concentra-tions is associated with the in-creased permeability of mitochon-drial membrane but not substantialloss of cytochrome c.Differential Effects of Ca2� on

Mitochondrial Membrane Po-tential (��m) in STHdhQ7/Q7

(Wild Type) and STHdhQ111/Q111(Mutant) Cells—To determine theeffects of Ca2� deregulation on��m, the ratiometric dye JC-1 wasused (38). In these experiments, iso-lated mitochondria were incubatedwith increasing Ca2� concentra-tions, keeping the same [mitochon-drial mass/Ca2� buffer volume]ratio as in the respiration experi-ments. Ca2� concentrations usedwere: 0 �M (EGTA-based respira-tion buffer), two low �M concentra-tions: 0.4 �M, 0.6 �M (software cal-culated) that correspond to 1.1 �M,2.2�M (Ca2� electrode determined)in Fig. 1B, at which decreases instate 3 respiration was observed,and 3 high �M concentrations: 150,500, and 1000 �M, where 150 �Mcorresponds approximately to theCa2� concentration at which we nolonger observed Ca2� uptake by thewild type mitochondria (Fig. 2).��m was also measured in Ca2�

uptake buffer (Cab), which is respiration buffer without EGTA,and contains �10 �M Ca2� as determined by using the Ca2�

electrode. ��m was determined as described under “Experi-mental Procedures.” FCCP was used to induce maximaldecrease of��m, as the positive control for the assay (Fig. 4). In

3 G. V. W. Johnson, unpublished observations.

FIGURE 3. Analysis of mitochondrial membrane integrity before and after Ca2� addition to mitochondriafrom STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, integrity of the mitochondria in wildtype and mutant cells. Representative respiration traces are shown to demonstrate the inability of exogenousNADH (5 mM) (inner mitochondrial membrane impermeable) to act as a respiratory substrate in mitochondriafrom both wild type and mutant cells. When pyruvate (10 mM) plus malate (10 mM) (pyr�mal) were used asrespiratory substrates, a significant increase in O2 consumption was observed upon ADP addition, while thiswas not the case when NADH was used. Numbers on the traces indicate O2 consumption rates. B, effects ofcytochrome c (cyt c) or cytochrome c plus NADH (cyt c�NADH) on respiration before and after Ca2� addition.Crude mitochondria were resuspended in respiration buffer without or with Ca2� (0.6 �M free Ca2� as calcu-lated by software), and respiration was measured as described under “Experimental Procedures.” Upon induc-tion of state 3 for 2 min (state 3), cytochrome c was added, and the rate measured for 2 min (state3�cyt c) andthen NADH was added and the rate measured for additional 2 min (state3�cyt c�NADH). In the absence ofCa2� neither cytochrome c nor cytochrome c plus NADH affected state 3 respiration. However, in the presenceof Ca2�, where significant decreases in state 3 rates were observed, cytochrome c plus NADH addition signif-icantly increased respiration. Cytochrome c alone did not affect state 3 rates in the presence of Ca2�. Data aremean S.E., n � 4 independent experiments. For statistical analyses, we used ANOVA with Tukey post test(*, p 0.05, compared with state 3 - Ca2�; #, p 0.05, compared with state 3 � Ca2�).



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the state 4 condition (ADP not added), we observed a Ca2�

concentration-dependent decrease of ��m. This decrease wassignificant in themutant at as low as 0.4�M, but in thewild typeonly at 500 �M Ca2� (Fig. 4A). The significant differencebetween the two could be observed at 0.6, Cab (�10 �M), 150,and 500 �M Ca2�, and was about 80% at 0.6 �M and 65% at 150�M. However, in the state 3 condition (ADP added) the signif-icant difference in ��m could be observed only starting at 150�M and was about 30% (Fig. 4B). These results show that ��mis significantly more sensitive to Ca2� in STHdhQ111/Q111

(mutant) compared with STHdhQ7/Q7 (wild type) cells. Also, thisdifference can be strongly attenuated by the presence of ADP.

Effects of Ca2� on H2O2 Production in Mitochondria fromSTHdhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant)Cells—Increased ROS production in mitochondria is usuallyassociated with perturbations of electron transfer in the oxi-dative phosphorylation process (39) and has been describedin conditions of increased mitochondrial membrane perme-ability (40). To assess the effects of Ca2� on ROS productionin the mitochondria from wild type and mutant cells theAmplex Red assay was used (28). This assay measures H2O2production and was carried out as described under “Experi-mental Procedures.” Isolated mitochondria were incubatedwith the increasing Ca2� concentrations, and H2O2 produc-tion was measured in the course of incubation. The Ca2�

concentrations used were as described for the ��m experi-ments (Fig. 4). In state 4, we observed Ca2� concentrationdependent increases in H2O2 that reached significance at1000 �M in wild type and at 500 and 1000 �M in the mutants(Fig. 5A). No differences at any of the data points wereobserved when wild type and mutants were compared (Fig.5A). In the state 3 condition (ADP added), we observedincreases in H2O2 production with Ca2� that reached signif-icance only in the mutant at 1000 �M Ca2� (Fig. 5B). Thetrend of increased H2O2 production with increasing Ca2�

concentrations was similar to what was observed for the��m change. At all Ca2� concentrations, mutant mitochon-dria displayed a greater increase in H2O2 production thanwild type (Fig. 5B). This increase was statistically significantat 0.4, 0.6, and 500 �M (Fig. 5B). In these experiments, rote-none treated mitochondria were used as an assay positivecontrol. As expected, in the wild type, rotenone treatmentcaused significant increases in H2O2 production. Interest-ingly, mutant mitochondria generated significantly lessH2O2 upon rotenone treatment when compared with thewild type.Effects of PTP Inhibitors on Mitochondrial Ca2� Uptake

Capacity in STHdhQ7/Q7 (Wild Type) and STHdhQ111/Q111(Mutant) Cells—Diminished Ca2� uptake capacity in themutant could be explained by a lower threshold for PTP open-ing in the mutant cells. To test this hypothesis, Ca2� uptakecapacity in the presence of PTP inhibitors was measured. Forthese experiments, cyclosporine A plus ADP plus oligomycinwas used, as this combination has been shown to be very effi-cient in inhibiting PTP in brain mitochondria (21). Addition ofPTP inhibitors increased mitochondrial Ca2� uptake capacityin both cell lines. This increase was greater and statisticallysignificant in the mutant mitochondria (ANOVA, Tukey posttest; p 0.01, n � 5 for mutant and n � 8 for mutant plus PTPinhibitors group) (Fig. 6). In the presence of PTP inhibitors,mitochondrial Ca2� uptake capacity reduction was stronglyattenuated in the mutant mitochondria, as the differencebetween the wild type and mutant mitochondria no longerreached statistical significance (Fig. 6). These results suggestthat STHdhQ111/Q111 (mutant) cells exhibit a lower thresholdfor PTP opening when compared with STHdhQ7/Q7 (wild type)cells.Effects of Ca2� on ��m in STHdhQ7/Q7 (Wild Type) and

STHdhQ111/Q111 (Mutant) Cells in Situ—Prior to evaluating theeffects of Ca2� on��m in situwithCM-H2TMRos andTMRE,

FIGURE 4. Differential effects of Ca2� on mitochondrial membrane poten-tial (��m) in state 4 and state 3 conditions in STHdhQ7/Q7 (wild type) andSTHdhQ111/Q111 (mutant) cells. A, effects of Ca2� on ��m in state 4. ��mwas measured in crude mitochondrial preparations using JC-1 as describedunder “Experimental Procedures.” In mutant cells ��m was significantlyreduced by as little as 0.4 �M Ca2� (software-calculated), whereas in wild type,only at 500 �M Ca2� was a decrease in ��m observed. Cab indicates meas-urements done in Ca2� uptake buffer (respiration buffer without EGTA). FCCPwas used as the positive control to induce a maximal decrease in ��m. Dataare mean S.E., n � 3 independent experiments. B, effects of Ca2� on ��min state 3. The presence of ADP (state 3) resulted in an attenuation of thedifferences between wild type and mutant observed in state 4 conditions.Data are mean S.E., n � 5 independent experiments. For statistical analyses,we used ANOVA followed by Tukey post test (*, p 0.05, compared with 0 �M

Ca2�) for each of the cell lines, and Student’s t test (#, p 0.05) to compare��m between the two cell lines at the different data points. Due to unequalvariances, Welch correction was applied for the comparison between 150 �M

points in state 4.



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control studies were carried out to validate the use of thesefluorescence dyes. These preliminary studies demonstratedthat treatment of both wild type or mutant cells with the mito-chondrial uncoupler FCCP resulted in a robust decrease inCM-H2TMRos and TMRE fluorescence, indicating a loss of��m (Fig. 7, A and B). To determine the effects of changes incytosolic Ca2� levels on ��m, cells were treated with a lowconcentration of the ionophore 4-BrA23187 (1 nM), which doesnot negatively impact cell viability (41). After treatment withthe ionophore for 5 min, 4 �mol of Ca2� additions were made

approximately every 5 min, to obtain the increasing concentra-tions of Ca2� in themedium (2, 4, 6mM).With increasing Ca2�

concentrations a decrease in ��m was observed as measuredwith both CM-H2TMRos (Fig. 7C) and TMRE (Fig. 7D), and itwas significantly more pronounced in mutant cells (Fig. 7, Cand D). The difference in ��m between wild type and mutantcells was significant starting at 2 mM Ca2� in the media (Fig. 7,C and D). This was in accordance with the results obtained inmitochondrial preparations as shown in Fig. 4.

DISCUSSION

In this study, we provide evidence for the first time thatmito-chondrial Ca2� handling defects in cells of striatal origin thatexpress endogenous levels of mutant huntingtin result inimpairment of respiration, which could contribute to neuronaldysfunction and death in HD. Treatment of isolatedmitochon-dria from mutant cells with increasing Ca2� concentrations,resulted in a significant decrease in state 3 respiration at lowerCa2� thanmitochondria fromwild typecells. Further, theCa2�-dependent decrease of ��m was significantly greater in themutant cells compared with the wild type cells. However, the��m defect was markedly attenuated in the presence of ADP.Additionally themitochondrial Ca2�uptake capacity inmutantcells was significantly lower than what was observed in mito-chondria from wild type cells, which was completely abolishedby the presence of PTP inhibitors. Taken together these datademonstrate that the presence of mutant huntingtin at physio-logically relevant levels results in impaired Ca2� handling by

FIGURE 5. Effects of Ca2� on H2O2 production in mitochondria from STH-dhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, H2O2 productionin state 4. H2O2 production was evaluated using the Amplex Red assay asdescribed under ”Experimental Procedures.“ Ca2�-induced increases in H2O2production and significance was reached at 1000 �M Ca2� in wild type and at500, 1000 �M Ca2� in mutant. Cab indicates measurements done in Ca2�

uptake buffer (respiration buffer without EGTA) Results are mean S.E., n �5 independent experiments. Statistical analyses were done using ANOVA fol-lowed by Tukey post test (*, p 0.05, compared with 0 �M Ca2� for each cellline). B, H2O2 production in state 3. Ca2�-induced increases in H2O2 produc-tion, and significance was reached only in mutant cells at 1000 �M Ca2�.When compared with wild type cells, H2O2 production upon Ca2� treatmentwas trending higher in mutant cells, reaching significance at 0.4, 0.6, and 500�M Ca2� points when a paired t test analysis was done. Rotenone (Rot) addi-tion was used in the assay as a positive control. Interestingly, in mutant cellsH2O2 production was significantly lower upon rotenone treatment comparedwith wild type. Results are mean S.E., n � 4 independent experiments.Statistical analyses were done using ANOVA followed by Tukey post test(*, p 0.05, compared with 0 �M Ca2� for each cell line), paired t test(#, p 0.05) to compare the two cell lines at different data points, andStudent’s t test (&, p 0.05) for rotenone treatment groups.

FIGURE 6. Effects of PTP inhibitors on mitochondrial Ca2� uptake capac-ity in STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. Mito-chondrial Ca2� uptake was measured as described under ”Experimental Pro-cedures.“ A combination of cyclosporine A plus ADP plus oligomycin wasused to inhibit PTP and was added to the mitochondrial suspension (1 mg ofprotein in a 2-ml volume) before Ca2� additions. Additions of 20, 40, or 80nmol of Ca2� were used in separate experiments but the regimen of Ca2�

additions was always the same for both cell types in a given experiment. Ourresults indicate that PTP inhibitors addition caused an increase in Ca2�

uptake capacity and this was significant for the mutant cells but not for thewild type cells. In the absence of PTP inhibitors, mutant cells had a signifi-cantly decreased Ca2� uptake capacity compared with the wild type cells. Inthe presence of cyclosporine A plus ADP plus oligomycin the differencebetween wild type and mutant was no longer significant. Data are mean S.E., n � 5 - 9 independent measurements per group. Statistical analysis wasdone using ANOVA followed by Tukey post test (* and #, p 0.05).



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mitochondria which negatively impacts their function andhence likely impairs proper neuronal function.Excitotoxicity has been suggested as a key mechanism that is

responsible for neurodegeneration inHD (42), and dysfunctionat the level of the mitochondria could be a mediator of thistoxicity. Mitochondrial dysfunction could result in an increasein the sensitivity of neurons to glutamate, leading to Ca2�

induced cellular dysfunction and eventually cell death (the roleofmitochondria in excitotoxicity is discussed in Refs. 43–45). Ithas been shown previously that decreases in oxidative phos-phorylation and state 3 rates are early events in excitotoxicity,and occur prior to the commitment to cell death (46). More-over, it has been shown that free�MCa2� concentrations causesignificant decreases in oxidative phosphorylation in isolatedmitochondria (29). To determine if mutant huntingtin altersmitochondrial response toCa2�, wemeasured respiration ratesin the presence of increasingCa2� concentrations inmitochon-

dria from wild type (STHdhQ7/Q7) and mutant huntingtin(STHdhQ111/Q111)-expressing cells. We observed that thedecrease in the state 3 rate (ADP phosphorylation rate)occurred at significantly lower Ca2� concentrations in mutantcells compared with wild type cells, suggesting an increasedsensitivity to Ca2�.Elevated mitochondrial Ca2� levels are usually associated

with the opening of PTP. However, the mechanism of the oxi-dative phosphorylation decrease due to elevated Ca2� levels isnot quite clear (discussed in Ref. 46). It has been suggested thatpermeabilization of the outer mitochondrial membrane leadsto activation of an alternative respiratory pathway, which uti-lizes exogenous NADH oxidation on the outer mitochondrialmembrane and translocation of subsequently reduced cyto-chrome c to the innermembranewhere it feeds into complex IV(34, 35). In our study, addition of NADH and cytochrome ccaused an increase in the state 3 rates in the presence of Ca2�,

FIGURE 7. Effects of the Ca2� increase in the mitochondrial potential functionality in STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells in situ.A, wild type and mutant cells were loaded with Mitotracker H2 ROS Red (200 nM) to measure changes in mitochondrial potential in response to 10 �M FCCPtreatment for 30 min. FCCP induces a significant decrease in the mitochondrial potential in both cell types. Data are mean S.E. n � 3 separate experiments.B, to corroborate the observations with Mitotracker H2 ROS Red, wild type and mutant cells were loaded with TMRE (100 nM) to measure changes in mitochon-drial potential in response to 10 �M FCCP and similar results were obtained. Data are mean S.E. n � 3 separate experiments. C, wild type (black bars) andmutant (gray bars) cells were exposed to 1 nM 4-BrA23187 in the presence of increasing Ca2� concentrations. Quantification of Mitotracker H2 ROS Redfluorescence, as relative units, shows significantly reduced mitochondrial potential in mutant cells pretreated with 4-BrA23187 (4-BrA) at each Ca2� concen-tration used in comparison to wild type cells (*, p 0.05: n � 3). Data are mean S.E. n � 3 separate experiments, (*, p 0.05 by non-paired Student’s t test).D, wild type and mutant cells were loaded with TMRE (100 nM), and fluorescence changes were measured in wild type (black bars), and mutant (gray bars) cellsexposed to 1 nM 4-BrA23187 in the presence of increasing Ca2� concentrations. Addition of 1 nM 4-BrA23187 plus different CaCl2 concentrations induced asignificant loss of mitochondrial potential in mutant cells (*, p 0.05: n � 3) (gray bars) in comparison to wild type cells (black bars). Data are mean S.E. n �3 independent experiments, (*, p 0.05 by non-paired Student’s t test).



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but not in the absence, and cytochrome c alone did not increasestate 3. These results indicate the Ca2�-induced decrease inoxidative phosphorylation is associated with increased perme-abilization of the mitochondrial membrane but not with sub-stantial loss of cytochrome c.

In this study respiration rates were measured in mito-chondria isolated from STHdhQ7/Q7 (wild type) and STH-dhQ111/Q111 (mutant) cells in KCl-based EGTA containing res-piration buffer, and no significant differences were observed. Inour previous study, respiration rates were measured in digito-nin-permeabilized cells, in a sucrose-based buffer withoutEGTA, and significant decreases in the state 3 rates wereobserved in STHdhQ111/Q111 (mutant) cells (18). In the light ofthe current findings, it is likely that the experimental conditionsused in our previous studywere permissive for themutant hun-tingtin-dependent mitochondrial defects. These conditionsresulted in the deficits observed in state 3 rates for the mutantcells. Indeed it is likely that the presence of free Ca2� in thebuffers (e.g. from the sucrose) likely resulted in an increase inmitochondrial membrane permeability to a greater extent inthe mutant cells than the wild type cells, which caused theobserved differences in respiration.It has been shown that huntingtin associates with the

outer mitochondrial membrane (47). We tested the integrityof outer mitochondrial membrane in basal conditions. Wefound that mitochondria from both cell lines have a goodouter mitochondrial membrane integrity as state 3 rates didnot increase in the presence of NADH and cytochrome c.The inner mitochondrial membrane was of good integrity aswell, because NADH (inner mitochondrial membraneimpermeable) did not work as respiratory substrate. Also,state 4 rates were comparable in the two cell lines, indicatingsimilar levels of inner membrane proton leakage. However, itis still possible that changes in the integrity of mitochondrialmembrane due to mutant huntingtin are quite subtle andcould not be detected with the methods we used.Mutant huntingtin-expressing cells (STHdhQ111/Q111) showed

markedly enhanced ��m reduction in response to increasingCa2� concentrations both in vitro and in situ. Mitochondrialdepolarization in response to Ca2� is caused by Ca2� uptakeitself (partial and reversible depolarization) and by opening ofthe PTP when Ca2� uptake capacity is exceeded (completedepolarization) (38). Because in our experiments ��m wasmeasured in the population ofmitochondria, determined��mvalues could indicate the portion of mitochondria undergoingPTP associated with complete depolarization or ��m levelspresent in the majority of mitochondria at specific Ca2� con-centrations. As previously described, ADP prevents PTP open-ing and stabilizes��mby themechanism that includes bindingand stabilization of adenine nucleotide translocator (ANT) inthe conformation that prevents PTP opening (48). The differ-ences in ��m reduction between wild type and mutant cellswere significantly attenuated in the presence of ADP. This sug-gested that differences in the threshold for PTP opening, signif-icantly contributed to ��mdifferences observed between wildtype and mutant cells.The reduction of��mcould be observed at as low as 0.4 and

0.6 �M Ca2� (concentrations at which decreases in oxidative

phosphorylation were observed) in the mutant but not in thewild type cells. Because respiration was first monitored in state4 followed by the induction of state 3, the observed differencesin the state 3 rates are likely caused by the differences in the��m before the state 3 was induced. Indeed, no differences in��mwere observed at 0.4 and 0.6 �M Ca2� when measured instate 3 conditions.ROS production could contribute to Ca2�-induced PTP

opening (discussed in Ref. 40). As described, determination ofreleased H2O2 is a common and the most reliable measure ofmitochondrial ROS production (39). When added to isolatedmitochondria, Ca2� caused a dose-dependent increase in therelease of H2O2. However, no significant difference in releasedH2O2, between wild type andmutant, was observed at differentCa2� concentrations (state 4). Therefore, the dramatic differ-ences in��mandCa2�uptake capacity observed betweenwildtype andmutant are likely not caused by differences in the ROSproduction. However, in the presence of ADP (state 3), weobserved higher levels of released H2O2 in mutant mitochon-dria, which reached significance at several data points.Increased ROS in the presence of ADP would likely contributeto more pronounced decrease in state 3 rates observed inmutant mitochondria in the presence of Ca2�. Unexpectedly,we observed reduced H2O2 release from mutant mitochondriaupon rotenone treatment. Our results suggest that ROS pro-duction is likely not the mechanism for reduced threshold forPTPopening in themutant cells. However, therewas a trend formitochondria from mutant cells to exhibit altered ROS home-ostasis compared with wild type (modest increase in the pres-ence of Ca2� and ADP; decrease in the presence of rotenone).

In this article we observed that mitochondria from mutantcells had reduced Ca2� uptake capacity compared with mito-chondria fromwild type cells. As suggested in multiple articles,decreases in mitochondrial Ca2� uptake capacity could becaused by a decreased threshold for PTPopening (7, 49). In fact,when mitochondria from mutant cells were treated with PTPinhibitors (cyclosporine A, ADP, oligomycin), the defect inCa2� uptake was almost abolished. Decreased mitochondrialCa2� uptake has already been reported in other HD models.Panov et al. (7, 13) reported decreased Ca2� uptake in mito-chondria from HD lymphoblast cell line, and brain mitochon-dria from full-length huntingtin transgenic mice (YAC72).Recently, attenuatedCa2� uptakewas reported inmusclemito-chondria fromR6/2mice (30). Interestingly, recombinant trun-cated mutant huntingtin resulted in significant mitochondrialswelling at lower Ca2� loads than truncated wild type proteinwhen added to isolated mouse liver mitochondria (47). Thissuggested that the mechanism of mutant huntingtin-inducedmitochondrial dysfunction is possibly through its direct effectson mitochondria.Using multiple mitochondrial functional assays, we dem-

onstrated a mitochondrial Ca2� handling defect in mutant(STHdhQ111/Q111) cells. Although Ca2� overload could bethe cause of the earlier PTP opening in the mutant mito-chondria, our data suggest that it is the opening of the PTP atlower Ca2� concentrations resulting in membrane depolar-ization rather than Ca2� overload. ROS could contribute thePTP opening, but it did not correlate with the significant



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changes in ��m observed in the mutant cells, suggesting adifferent mechanism. Based on these and other findings, wehypothesize that the increased sensitivity of mutant mitochon-dria to increases in intracellular Ca2� results in depolarization,and this is an upstream event in the pathological cascade fol-lowed by decreases in respiration and Ca2� buffering capacity.In the light of these current findings, successful PTP inhibitionwould be beneficial, but, as already discussed in terms of exci-totoxicity (38), limiting mitochondrial Ca2� uptake by manip-ulating its setpoint (throughCa2�-uniporter inhibition orNa�-Ca2�-exchanger activation) is also likely to be beneficial in thetreatment of HD.

Acknowledgment—We thank Dr. P. Bernardi for suggestions andadvice regarding outer mitochondrial membrane integrityexperiments.

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Tamara Milakovic, Rodrigo A. Quintanilla and Gail V. W. Johnsonin Clonal Striatal Cells: FUNCTIONAL CONSEQUENCES

Mutant Huntingtin Expression Induces Mitochondrial Calcium Handling Defects

doi: 10.1074/jbc.M603845200 originally published online September 13, 20062006, 281:34785-34795.J. Biol. Chem.

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http://www.jbc.org/content/281/46/34785.full.html#ref-list-1

http://www.jbc.org/

Documents

Mutant Huntingtin Expression Induces Mitochondrial Calcium