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Originally published In Press as doi:10.1074/jbc.M603845200 on September 13, 2006

J. Biol. Chem., Vol. 281, Issue 46, 34785-34795, November 17, 2006
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Mutant Huntingtin Expression Induces Mitochondrial Calcium Handling Defects in Clonal Striatal Cells

FUNCTIONAL CONSEQUENCES*

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

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

Received for publication, April 21, 2006 , and in revised form, August 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington disease (HD) is caused by a pathological elongation of CAG repeats in the huntingtin protein gene and is characterized by atrophy and neuronal loss primarily in the striatum. Mitochondrial dysfunction and impaired Ca2+ homeostasis in HD have been suggested previously. Here, we elucidate the effects of Ca2+ on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) huntingtin-expressing cells of striatal origin. When treated with increasing Ca2+ concentrations, mitochondria from mutant huntingtin-expressing cells showed enhanced sensitivity to Ca2+, as they were more sensitive to Ca2+-induced decreases in state 3 respiration and {Delta}{Psi}m, than mitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2+ uptake capacity in comparison with wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The {Delta}{Psi}m defect was attenuated in the presence of ADP and the decreases in Ca2+ uptake capacity were abolished in the presence of Permeability Transition Pore (PTP) inhibitors. These findings clearly indicate that mutant huntingtin-expressing cells have mitochondrial Ca2+ handling defects that result in respiratory deficits and that the increased sensitivity to Ca2+ induced mitochondrial permeabilization maybe a contributing mechanism to the mitochondrial dysfunction in HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington disease (HD)2 is a neurodegenerative disease that is inherited in an autosomal dominant manner. It belongs to a family of CAG expansion diseases and is caused by the pathological elongation of the CAG repeats in exon one of the huntingtin protein gene (1). Symptoms and disease progression are caused by dysfunction and loss of neurons starting in the striatum (specifically medium spiny neurons), but progressing to cortex and to a lesser extent to other brain regions in the later stages of the disease (2). Disease is caused by the toxic gain of function of mutant protein but some loss of function may also contribute to the pathogenesis (for review see Ref. 3). The toxic gain of function of mutant huntingtin has not been clearly defined, but there are findings suggesting that mutant huntingtin causes transcriptional dysregulation (4), ubiquitin-proteasome system dys-function (5), Ca2+ homeostasis dysfunction (6, 7), and mitochondrial dysfunction (7–10).

Mitochondrial dysfunction in HD has been suggested primarily by the studies showing impairment of mitochondrial complexes (II, III, and IV) specifically in the striatum in the late stages HD patients (8–10). Administration of the mitochondrial complex II inhibitor 3-nitropropionic (3-NP) in both rodents and nonhuman primates resulted in symptoms and neuropathology that resemble HD (11, 12). Further, studies have shown impairment of mitochondrial Ca2+ buffering in HD lymphoblast cell line and brain mitochondria 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). Previously it has been demonstrated that mitochondrial dysfunction can lead to neuronal sensitization to glutamate leading to excitotoxic cellular dysfunction and cell death (14, 15). Hence, even though HD is not a classic mitochondrial disease (16), elucidation of mitochondrial dysfunction mechanisms would likely provide important insight in HD pathogenesis.

To study the effects of mutant huntingtin on mitochondrial function, conditionally immortalized cells of striatal origin that express endogenous, comparable levels of either wild type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin were used (17). These cell lines are prepared from wild type (HdhQ7/Q7) and mutant huntingtin knock-in mice (HdhQ111/Q111) (17) and therefore the STHdhQ111/Q111 cell line is a genetically accurate cell model of HD. In our previous study (18), we investigated the effects of mutant huntingtin on mitochondrial electron transport chain complexes using STHdhQ7/Q7 and STHdhQ111/Q111 cell lines. Given the fact that the metabolic thresholds and enzyme activities of electron transport chain complexes were not different between the two cell lines, it is likely that the mitochondrial complex deficits are a later event in the course of HD pathogenesis, indeed in low grade HD cases no deficits in the enzyme activities of electron transport chain complexes were observed (19).

In the present study, we examined the effects of Ca2+ on mitochondria from STHdhQ7/Q7 and STHdhQ111/Q111 cells. Isolated mitochondria were treated with increasing Ca2+ concentrations and mitochondrial function was assessed using different assays. We determined that mutant huntingtin-expressing cells have decreased Ca2+ uptake capacity, and exhibit increased sensitivity to Ca2+-induced decreases in respiration and {Delta}{Psi}m. The {Delta}{Psi}m defect was attenuated in the presence of ADP and the decrease in Ca2+ uptake capacity was abolished in the presence of Permeability Transition Pore (PTP) inhibitors. This study clearly demonstrates that mitochondrial Ca2+ buffering capacity in STHdhQ111/Q111 cells is compromised, and suggests increased sensitivity to Ca2+-induced mitochondrial permeabilization as a mechanism of mitochondrial dysfunction in HD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were from Sigma-Aldrich unless otherwise noted. All buffers used in experiments with crude mitochondrial 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 expressing endogenous wild type huntingtin and the homozygous mutant STHdhQ111/Q111 cell line expressing comparable levels of mutant huntingtin with 111 glutamines were used. Cell lines were prepared from wild type mice and homozygous HdhQ111/Q111 knock-in mice and were described previously (17). Culturing conditions were the same as described in our previous study (18).

Isolation of Mitochondria—Cells were grown on 150-mm plates until {approx} 80–90% confluency, washed twice with cavitation buffer (250 mM sucrose, 5 mM HEPES, 3 mM MgCl2, 1 mM EGTA, pH 7.3 corrected with 5 M KOH) and scraped into cavitation buffer using soft rubber scrapers. Cells were opened using N2 cavitation for 5 min at 250 psi on ice, and samples were additionally homogenized with 1 stroke in a glass Dounce homogenizer. Homogenates were centrifuged at 7000 x g for 10 min at 4 °C. Supernatants were aspirated, and pellets were resuspended in cavitation buffer and used as crude mitochondrial preparations. Protein concentrations in crude mitochondrial preparations were determined using the bicinchoninic acid assay (Pierce), and aliquots were then prepared that contained the indicated protein content for each measurement. Aliquots were centrifuged at 7000 x g for 10 min and kept on ice in cavitation buffer until use in each assay.

Measurement of Mitochondrial Respiration—Respiration rates were measured using an oxygraph (Hansatech Instruments) as described previously (18). Crude mitochondrial preparations (0.5-mg aliquots) were resuspended in respiration buffer (130 mM KCl, 20 mM HEPES, 2 mM MgCl2, 2 mM EGTA, 2 mM potassium phosphate (KH2PO4/K2HPO4, 1:1.78), 1% essentially fatty acids free bovine serum albumin, pH 7.2 adjusted with 5 M KOH) to a final concentration of 1 mg/ml. The mitochondrial suspension (0.5 ml volume) was placed in the respiratory chamber and allowed to equilibrate for 2 min. Respiratory substrate (glutamate (10 mM) plus malate (10 mM) or succinate (5 mM) with rotenone (10 µM)) was then added and state 4 respiration was measured for 2 min, ADP (1.5 mM) was then added, and state 3 respiration was measured for a further 2–4 min. Rates were normalized to citrate synthase activity in the same samples. Citrate synthase activity was determined as previously described (18).

Ca2+ Titration Experiments—Respiration buffers containing specific free Ca2+ concentrations (Ca2+-EGTA respiration buffers) were prepared on the day of the experiment. To calculate the amount of total Ca2+ that was needed to achieve the appropriate free Ca2+ concentration in the respiration buffer that contained 2 mM EGTA we used MaxChelator software (20). Each Ca2+-EGTA respiration buffer was prepared separately by diluting each specific 100x CaCl2 stock in the respiration buffer and correcting its pH to 7.2 using 0.1 M KOH in the respiration buffer. CaCl2 stocks were prepared from CaCl2·2H2O (minimum 99%), that was dried overnight and stored in a desiccation chamber until use. Crude mitochondrial preparations were dissolved in prepared Ca2+-EGTA respiration buffers, and respiration rates were measured as described above. The period between buffer addition to the mitochondrial preparation and initiation of state 3 was ~5 min.

Free Ca2+ concentrations in the Ca2+-EGTA buffers were checked using a calibrated Ca2+ electrode on the day of the experiment. Measured concentrations were averaged and presented on the X-axis of Ca2+ titration experiments graphs. Actually concentrations were always slightly higher than those calculated by software.

Cytochrome c and NADH Respiration Experiments—Respiration experiments were performed as described above. State 3 respiration was measured for 2 min prior to the addition of cytochrome c (30 µM), and respiration was monitored for another 2 min. This was followed by the addition of NADH (5 mM), 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 preparation was resuspended in Ca2+ uptake buffer (130 mM KCl, 20 mM HEPES, 2 mM MgCl2, 2 mM potassium phosphate (KH2PO4/K2HPO4, 1:1.78), 1% bovine serum albumin, pH 7.2 adjusted with 5 M KOH) and placed in the oxygraph respiratory chamber. The respiratory chamber was thermostatted at 37 °C, and its contents were constantly mixed with an electromagnetic stirrer bar. Glutamate (10 mM) and malate (10 mM) were added as respiratory substrates. Ca2+ and reference electrodes were added to the chamber from the top. Starting volume of the reaction was 2 ml. The chamber was kept open during an experiment. Ca2+ additions were performed using fine tubing and a Hamilton syringe. 5, 10, and 20 mM CaCl2 stocks were used to make 10, 20, 40, or 80 nmol of Ca2+ additions. The Ca2+ electrode measures extramitochondrial Ca2+ and increases in the signal present as downward deflections on the traces.

To observe the effects of PTP inhibition on Ca2+ uptake capacity we used cyclosporine A (1 µM) plus ADP (50 µM) plus oligomycin (2 µg/ml). The PTP inhibitors were added to the respiratory chamber prior to Ca2+ additions (21).

To calculate Ca2+ uptake capacity, we counted number of Ca2+ additions until the addition after which no uptake was observed (trace horizontal). The number of additions was multiplied by the nmol of Ca2+ per addition, and normalized to protein content.

Mitochondrial Membrane Potential ({Delta}{Psi}m) Determination in Live Cells—Mitochondrial membrane potential was estimated using the specific mitochondrial probes: Mitotracker Red (CM-H2TMRos) and tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes) (22–26). Cells were grown on poly-L-lysine-coated plates and cultured for 3 days. The cells were then loaded for 30 min with CM-H2TMRos in KRH-glucose, washed, and allowed to equilibrate for 15 min. Cell plates were then mounted in a chamber on the stage of a confocal laser scanning microscope (Leica model TCS SP2). Quantitative measurements of CM-H2TMRos fluorescence were performed by confocal microscopy (Leica model TCS SP2), using a 40x water immersion lens. CM-H2TMRos fluorescence images were obtained by excitation at 563 nm, reflection off a dichroic mirror with a cut-off wavelength at 564 nm, and longpass emission filtering at 590 nm. For TMRE experiments, cells were loaded with TMRE (100 nM) for 45 min in KRH-glucose, and then were mounted on the stage for confocal microscopy. TMRE fluorescence was detected exciting with a 561 nm He-Ne laser line very heavily attenuated (10% laser power), and the emission was collected at >563 nm (22). Signal from control cells and cells treated with different stimuli were compared using identical settings for laser power, confocal thickness, and detector sensitivity for each dye and separate experiment (23, 25, 26). The images were analyzed with LCS Leica confocal software and recorded as the mean Mitotracker Red or TMRE fluorescence signal per live cell.

Measurement of Mitochondrial Membrane Potential ({Delta}{Psi}m) in Mitochondrial Preparations—{Delta}{Psi}m was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) according to a published protocol with modifications (27). Modifications were made so that method could be used with isolated mitochondria. To measure {Delta}{Psi}m at different Ca2+ concentrations, crude mitochondrial preparation was aliquoted into the wells of 96-well plate (50 µg/well). The plate was centrifuged at 3220 x g for 10 min at 4 °C, and supernatants were carefully aspirated. Ca-EGTA respiration buffers with 0, 0.4, or 0.6 µM (software calculated) free Ca2+ or Ca2+ uptake buffer with 150, 500, or 1000 µM Ca2+ each supplemented with glutamate (10 mM), malate (10 mM), and with (for state 3) or without (for state 4) ADP (1.5 mM) were added to separate wells in duplicates (50 µl/well) and incubated at 37 °C for 10 min. Supernatants were carefully aspirated, and the same buffers but containing JC-1 (5 µg/ml) were added to the wells (50 µl/well). Wells in which FCCP (20 µM) was also added were considered as positive controls. The plate was incubated for 30 min, at 37 °C in dark, supernatants were aspirated, and fluorescence was read at 485/528 nm and 530/590 nm. The ratio between the fluorescence was used to describe {Delta}{Psi}m as published previously (27).

Measurement of Mitochondrial H2O2 Production—To determine mitochondrial ROS production we used an Amplex Red (Molecular Probes) assay (28). Crude mitochondrial preparations were aliquoted (100 µg/well) and pelleted onto a 96-well plate as described for measuring {Delta}{Psi}m. Mitochondrial pellets were covered with Ca-EGTA respiration buffers with 0, 0.4, or 0.6 µM (software calculated) free Ca2+ or Ca2+ uptake buffer with 150, 500, or 1000 µM Ca2+ each supplemented with glutamate (10 mM), malate (10 mM), Amplex Red (50 µM), horseradish peroxidase (0.01 units/ml or 0.1 units/ml), and with (for state 3) or without (for state 4) ADP (1.5 mM). Plate was read in the kinetic mode for 30 min at excitation/emission wavelengths 530/590 nm at 37 °C. Rates of H2O2 production were determined using a standard curve.

Statistical Analysis—Results were analyzed using ANOVA, Student's t test, or paired t test as indicated. Differences were considered significant if p ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Ca2+ on Respiration in Mitochondria from STH-dhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant) Cells—It has been shown previously that at the free concentrations higher than 1 µM, Ca2+ causes strong inhibition of oxidative phosphorylation (29). To determine the effects of Ca2+ on oxidative phosphorylation in mitochondria isolated from the cells expressing endogenous levels of wild type (STHdhQ7/Q7) or mutant (STHdhQ111/Q111) huntingtin, we measured state 4 and state 3 respiration rates in respiration buffers containing increasing free µM Ca2+ concentrations. In these experiments, EGTA-based respiration buffer was used for 0 µM Ca2+ and Ca2+-EGTA respiration buffers were prepared as described under "Experimental Procedures." At 0 µM Ca2+, we observed no differences in the state 4 or state 3 respiration rates between wild type and mutant cells when glutamate plus malate (complex I substrate) or succinate (complex II substrate) were used as substrates (Fig. 1A). As described earlier (29), with increasing free µM Ca2+ concentrations decreases in the state 3 rates were observed (Fig. 1B). However, this decrease was more pronounced in the mitochondria from the mutant huntingtin-expressing cells, reaching significance at lower Ca2+ concentrations than in the wild type (Fig. 1B). State 4 rates increased with increasing Ca2+ concentrations, reaching significance only in the mutant at the highest Ca2+ concentration used (Fig. 1B). To describe overall changes in the respiration rates, we calculated Respiratory Control Ratios (RCRs) at the different Ca2+ concentrations. RCR was calculated as the ratio between state 3 and state 4 rates. A decrease in RCR was observed with increasing Ca2+ concentrations and was more pronounced in mitochondria from mutant cells, reaching significance at the lower Ca2+ concentrations than in the wild type cells (Fig. 1C). These results indicate that mitochondria from STHdhQ111/Q111 (mutant) cells are more sensitive to Ca2+-induced changes in oxidative 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—Several studies have suggested that there is reduced mitochondrial Ca2+ buffering capacity in HD. Panov et al. (7, 13) demonstrated diminished Ca2+ uptake capacity in mitochondria from HD lymphoblast cell lines, and brain mitochondria from the full-length mutant huntingtin-overexpressing mice (YAC72) (7), whereas others demonstrated diminished Ca2+ uptake in muscle mitochondria from R6/2 mice (30). To comprehensively describe the effects of Ca2+ on mitochondria in our model, we determined mitochondrial Ca2+ uptake capacity in STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. For these experiments we used a Ca2+-sensitive electrode, as described under "Experimental Procedures." To determine, mitochondrial Ca2+ uptake, isolated mitochondria (1.5 mg/2 ml) were placed in a 37 °C thermostatted chamber and challenged with 10-nmol Ca2+ pulses every 3 min. Representative traces are shown in Fig. 2C. Ca2+ uptake capacity was calculated as described under "Experimental Procedures." We observed that mitochondria from STHdhQ111/Q111 (mutant) cells have significantly diminished Ca2+ uptake capacity compared with mitochondria from STHdhQ7/Q7 (wild type) cells (Fig. 2A). To determine the "initial uptake" rates we calculated the average of the rates after the second, third, and fourth additions of Ca2+ and determined that the "initial uptake" rates were significantly diminished in the mitochondria from the mutant cells (Fig. 2B). These results indicate that mitochondria from STHdhQ111/Q111 (mutant) cells have a Ca2+-buffering defect, as they can take up less Ca2+ than the mitochondria from wild type cells. Because ER contamination of the mitochondrial preparation was a possibility, we confirmed the mitochondrial nature of the Ca2+ uptake in our mitochondrial preparations, as the addition of uncoupler (FCCP) caused release of Ca2+, and pretreatment of the cells with thapsigargin (which blocks the Ca2+ uptake pump of the ER Ref. 31) did not produce any change in the Ca2+ uptake capacity (Fig. 2D).


Figure 1
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  		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 were incubated with either glutamate plus malate (glu.+mal.) or succinate with rotenone (succ.+rot.) as respiratory substrates, and respiration rates were determined as described under "Experimental Procedures." Rates were normalized to citrate synthase activity measured in the same samples. No significant differences were observed. B, respiration rates measured in the presence of increasing free Ca2+ concentrations. Crude mitochondria were resuspended in Ca-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 µM free Ca2+ concentrations were observed, with the decreases occurring at the lower Ca2+ concentrations in mitochondria 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+ concentrations 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 the Tukey post test (*, p < 0.05) was used.

 


Figure 2
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  		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 given 10-nmol Ca2+ pulses every 3 min, until uptake could not be observed anymore. Ca2+ uptake capacities were calculated by multiplying the number of pulses by 10 nmol and normalizing it to mg of protein. Significantly lower Ca2+ uptake capacity in mitochondria from mutant cells as compared with mitochondria from 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 fourth additions of Ca2+. Initial Ca2+ uptake rates were significantly lower in mitochondria from mutant cells compared with wild type cells. Data are mean ± S.E. of three 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 preparation. Mitochondria from mutant cells were used in these control experiments. Addition of FCCP caused release of Ca2+ from mitochondria as evident by increase in [Ca2+] in the reaction buffer (downward deflection of the trace after FCCP). Thapsigargin (1 µM) pretreatment of the cells for 30 min did not affect Ca2+ uptake capacity.

 
Analysis of Mitochondrial Membrane Integrity Before and After Ca2+ Addition in STHdhQ7/Q7 (Wild Type) and STH-dhQ111/Q111 (Mutant) Cells—Ca2+ overload of mitochondria results in increased mitochondrial membrane permeability (32). To further study the cause of differences between mitochondria from wild type and mutant cells in their sensitivity to Ca2+, we wanted to determine if the decrease in respiration observed in the presence of free µM Ca2+ concentrations was associated with increased permeability of the mitochondrial membrane. First, we analyzed the integrity of mitochondrial membrane in the basal conditions. NADH is the substrate for mitochondrial complex I. However, the inner mitochondrial membrane is not permeable to exogenous NADH (33). When pyruvate plus malate was added to provide reduced adenine dinucleotides (NADH, FADH2) inside the mitochondria we observed significant state 3 rates upon ADP addition (Fig. 3A). However, when NADH was used as the respiratory substrate, we did not observe induction of state 3 respiration in mitochondria from any of the cell lines (Fig. 3A). Representative traces are shown (Fig. 3A). This indicates good integrity of mitochondrial inner membrane in basal conditions in mitochondria from both cell lines.

It has been suggested that damage of the outer mitochondrial membrane results in the activation of alternative respiratory pathway in the presence of exogenous NADH and cytochrome c (34–37). In this pathway, NADH is oxidized at the outer mitochondrial membrane leading to reduction of exogenous cytochrome c. As described previously, if outer membrane is being compromised, cytochrome c will translocate to the complex IV and stimulate respiration (35). To assess mitochondrial membrane integrity, we measured the effects of NADH and cytochrome c on state 3 respiration in the absence or presence of Ca2+. For these experiments, we chose Ca2+ concentration that caused a significant decrease in state 3 rate in the mitochondria from both cell lines. Experiments were carried out as described under "Experimental Procedures." In the absence of Ca2+, neither cytochrome c alone nor cytochrome c plus NADH had any effect on respiration (Fig. 3B). This indicated good integrity of the outer mitochondrial membrane in both cell lines. However in the presence of Ca2+, when the state 3 respiration was decreased, cytochrome c plus NADH caused a significant increase in state 3 (Fig. 3B). Alamethicin, forms pores in the membrane, was used as a positive control for the method (not shown).3 Cytochrome c alone did not affect state 3 respiration in the presence of Ca2+ (Fig. 3B), suggesting that no substantial loss of cytochrome c is causing the decrease in state 3 rate. These results suggest that mitochondria from both wild type and mutant cells do have good membrane integrity in the absence of Ca2+. Further, the decrease in state 3 rates in the presence of µM Ca2+ concentrations is associated with the increased permeability of mitochondrial membrane but not substantial loss of cytochrome c.


Figure 3
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  		FIGURE 3.
Analysis of mitochondrial membrane integrity before and after Ca2+ addition to mitochondria from STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, integrity of the mitochondria in wild type and mutant cells. Representative respiration traces are shown to demonstrate the inability of exogenous NADH (5 mM) (inner mitochondrial membrane impermeable) to act as a respiratory substrate in mitochondria from both wild type and mutant cells. When pyruvate (10 mM) plus malate (10 mM) (pyr+mal) were used as respiratory substrates, a significant increase in O2 consumption was observed upon ADP addition, while this was not the case when NADH was used. Numbers on the traces indicate O2 consumption rates. B, effects of cytochrome 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 µM free Ca2+ as calculated by software), and respiration was measured as described under "Experimental Procedures." Upon induction of state 3 for 2 min (state 3), cytochrome c was added, and the rate measured for 2 min (state3+cyt c) and then NADH was added and the rate measured for additional 2 min (state3+cyt c+NADH). In the absence of Ca2+ neither cytochrome c nor cytochrome c plus NADH affected state 3 respiration. However, in the presence of Ca2+, where significant decreases in state 3 rates were observed, cytochrome c plus NADH addition significantly increased respiration. Cytochrome c alone did not affect state 3 rates in the presence of Ca2+. Data are mean ± 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+).

 
Differential Effects of Ca2+ on Mitochondrial Membrane Potential ({Delta}{Psi}m) in STHdhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant) Cells—To determine the effects of Ca2+ deregulation on {Delta}{Psi}m, the ratiometric dye JC-1 was used (38). In these experiments, isolated mitochondria were incubated with increasing Ca2+ concentrations, keeping the same [mitochondrial mass/Ca2+ buffer volume] ratio as in the respiration experiments. Ca2+ concentrations used were: 0 µM (EGTA-based respiration buffer), two low µM concentrations: 0.4 µM, 0.6 µM (software calculated) that correspond to 1.1 µM, 2.2 µM (Ca2+ electrode determined) in Fig. 1B, at which decreases in state 3 respiration was observed, and 3 high µM concentrations: 150, 500, and 1000 µM, where 150 µM corresponds approximately to the Ca2+ concentration at which we no longer observed Ca2+ uptake by the wild type mitochondria (Fig. 2). {Delta}{Psi}m was also measured in Ca2+ uptake buffer (Cab), which is respiration buffer without EGTA, and contains ~10 µM Ca2+ as determined by using the Ca2+ electrode. {Delta}{Psi}m was determined as described under "Experimental Procedures." FCCP was used to induce maximal decrease of {Delta}{Psi}m, as the positive control for the assay (Fig. 4). In the state 4 condition (ADP not added), we observed a Ca2+ concentration-dependent decrease of {Delta}{Psi}m. This decrease was significant in the mutant at as low as 0.4 µM, but in the wild type only at 500 µM Ca2+ (Fig. 4A). The significant difference between the two could be observed at 0.6, Cab (~10 µM), 150, and 500 µM Ca2+, and was about 80% at 0.6 µM and 65% at 150 µM. However, in the state 3 condition (ADP added) the significant difference in {Delta}{Psi}m could be observed only starting at 150 µM and was about 30% (Fig. 4B). These results show that {Delta}{Psi}m is significantly more sensitive to Ca2+ in STHdhQ111/Q111 (mutant) compared with STHdhQ7/Q7 (wild type) cells. Also, this difference can be strongly attenuated by the presence of ADP.


Figure 4
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  		FIGURE 4.
Differential effects of Ca2+ on mitochondrial membrane potential ({Delta}{Psi}m) in state 4 and state 3 conditions in STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, effects of Ca2+ on {Delta}{Psi}m in state 4. {Delta}{Psi}m was measured in crude mitochondrial preparations using JC-1 as described under "Experimental Procedures." In mutant cells {Delta}{Psi}m was significantly reduced by as little as 0.4 µM Ca2+ (software-calculated), whereas in wild type, only at 500 µM Ca2+ was a decrease in {Delta}{Psi}m observed. Cab indicates measurements done in Ca2+ uptake buffer (respiration buffer without EGTA). FCCP was used as the positive control to induce a maximal decrease in {Delta}{Psi}m. Data are mean ± S.E., n = 3 independent experiments. B, effects of Ca2+ on {Delta}{Psi}m in state 3. The presence of ADP (state 3) resulted in an attenuation of the differences 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 µM Ca2+) for each of the cell lines, and Student's t test (#, p < 0.05) to compare {Delta}{Psi}m between the two cell lines at the different data points. Due to unequal variances, Welch correction was applied for the comparison between 150 µM points in state 4.

 
Effects of Ca2+ on H2O2 Production in Mitochondria from STHdhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant) Cells—Increased ROS production in mitochondria is usually associated with perturbations of electron transfer in the oxidative phosphorylation process (39) and has been described in conditions of increased mitochondrial membrane permeability (40). To assess the effects of Ca2+ on ROS production in the mitochondria from wild type and mutant cells the Amplex Red assay was used (28). This assay measures H2O2 production and was carried out as described under "Experimental Procedures." Isolated mitochondria were incubated with the increasing Ca2+ concentrations, and H2O2 production was measured in the course of incubation. The Ca2+ concentrations used were as described for the {Delta}{Psi}m experiments (Fig. 4). In state 4, we observed Ca2+ concentration dependent increases in H2O2 that reached significance at 1000 µM in wild type and at 500 and 1000 µM in the mutants (Fig. 5A). No differences at any of the data points were observed when wild type and mutants were compared (Fig. 5A). In the state 3 condition (ADP added), we observed increases in H2O production with Ca2+2 that reached significance only in the mutant at 1000 µM Ca2+ (Fig. 5B). The trend of increased H2O2 production with increasing Ca2+ concentrations was similar to what was observed for the {Delta}{Psi}m change. At all Ca2+ concentrations, mutant mitochondria displayed a greater increase in H2O2 production than wild type (Fig. 5B). This increase was statistically significant at 0.4, 0.6, and 500 µM (Fig. 5B). In these experiments, rotenone treated mitochondria were used as an assay positive control. As expected, in the wild type, rotenone treatment caused significant increases in H2O2 production. Interestingly, mutant mitochondria generated significantly less H2O2 upon rotenone treatment when compared with the wild 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 the mutant could be explained by a lower threshold for PTP opening in the mutant cells. To test this hypothesis, Ca2+ uptake capacity in the presence of PTP inhibitors was measured. For these experiments, cyclosporine A plus ADP plus oligomycin was used, as this combination has been shown to be very efficient in inhibiting PTP in brain mitochondria (21). Addition of PTP inhibitors increased mitochondrial Ca2+ uptake capacity in both cell lines. This increase was greater and statistically significant in the mutant mitochondria (ANOVA, Tukey post test; p < 0.01, n = 5 for mutant and n = 8 for mutant plus PTP inhibitors group) (Fig. 6). In the presence of PTP inhibitors, mitochondrial Ca2+ uptake capacity reduction was strongly attenuated in the mutant mitochondria, as the difference between the wild type and mutant mitochondria no longer reached statistical significance (Fig. 6). These results suggest that STHdhQ111/Q111 (mutant) cells exhibit a lower threshold for PTP opening when compared with STHdhQ7/Q7 (wild type) cells.

Effects of Ca2+ on {Delta}{Psi}m in STHdhQ7/Q7 (Wild Type) and STHdhQ111/Q111 (Mutant) Cells in Situ—Prior to evaluating the effects of Ca2+ on {Delta}{Psi}m in situ with CM-H2TMRos and TMRE, control studies were carried out to validate the use of these fluorescence dyes. These preliminary studies demonstrated that treatment of both wild type or mutant cells with the mitochondrial uncoupler FCCP resulted in a robust decrease in CM-H2TMRos and TMRE fluorescence, indicating a loss of {Delta}{Psi}m (Fig. 7, A and B). To determine the effects of changes in cytosolic Ca2+ levels on {Delta}{Psi}m, cells were treated with a low concentration of the ionophore 4-BrA23187 (1 nM), which does not negatively impact cell viability (41). After treatment with the ionophore for 5 min, 4 µmol of Ca2+ additions were made approximately every 5 min, to obtain the increasing concentrations of Ca2+ in the medium (2, 4, 6 mM). With increasing Ca2+ concentrations a decrease in {Delta}{Psi}m was observed as measured with both CM-H2TMRos (Fig. 7C) and TMRE (Fig. 7D), and it was significantly more pronounced in mutant cells (Fig. 7, C and D). The difference in {Delta}{Psi}m between wild type and mutant cells was significant starting at 2 mM Ca2+ in the media (Fig. 7, C and D). This was in accordance with the results obtained in mitochondrial preparations as shown in Fig. 4.


Figure 5
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  		FIGURE 5.
Effects of Ca2+ on H2O2 production in mitochondria from STH-dhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. A, H2O2 production in state 4. H2O2 production was evaluated using the Amplex Red assay as described under"Experimental Procedures."Ca2+-induced increases in H2O2 production and significance was reached at 1000 µM Ca2+ in wild type and at 500, 1000 µM 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 followed by Tukey post test (*, p < 0.05, compared with 0 µM Ca2+ for each cell line). B, H2O2 production in state 3. Ca2+-induced increases in H2O2 production, and significance was reached only in mutant cells at 1000 µM Ca2+. When compared with wild type cells, H2O2 production upon Ca2+ treatment was trending higher in mutant cells, reaching significance at 0.4, 0.6, and 500 µM Ca2+ points when a paired t test analysis was done. Rotenone (Rot) addition was used in the assay as a positive control. Interestingly, in mutant cells H2O2 production was significantly lower upon rotenone treatment compared with 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 µM Ca2+ for each cell line), paired t test (#, p < 0.05) to compare the two cell lines at different data points, and Student's t test (&, p < 0.05) for rotenone treatment groups.

 


Figure 6
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  		FIGURE 6.
Effects of PTP inhibitors on mitochondrial Ca2+ uptake capacity in STHdhQ7/Q7 (wild type) and STHdhQ111/Q111 (mutant) cells. Mitochondrial Ca2+ uptake was measured as described under"Experimental Procedures."A combination of cyclosporine A plus ADP plus oligomycin was used to inhibit PTP and was added to the mitochondrial suspension (1 mg of protein in a 2-ml volume) before Ca2+ additions. Additions of 20, 40, or 80 nmol 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. Our results indicate that PTP inhibitors addition caused an increase in Ca2+ uptake capacity and this was significant for the mutant cells but not for the wild type cells. In the absence of PTP inhibitors, mutant cells had a significantly decreased Ca2+ uptake capacity compared with the wild type cells. In the presence of cyclosporine A plus ADP plus oligomycin the difference between wild type and mutant was no longer significant. Data are mean ± S.E., n = 5 - 9 independent measurements per group. Statistical analysis was done using ANOVA followed by Tukey post test (* and #, p < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide evidence for the first time that mitochondrial Ca2+ handling defects in cells of striatal origin that express endogenous levels of mutant huntingtin result in impairment of respiration, which could contribute to neuronal dysfunction and death in HD. Treatment of isolated mitochondria from mutant cells with increasing Ca2+ concentrations, resulted in a significant decrease in state 3 respiration at lower Ca2+ than mitochondria from wild type cells. Further, the Ca2+-dependent decrease of {Delta}{Psi}m was significantly greater in the mutant cells compared with the wild type cells. However, the {Delta}{Psi}m defect was markedly attenuated in the presence of ADP. Additionally the mitochondrial Ca2+ uptake capacity in mutant cells was significantly lower than what was observed in mitochondria from wild type cells, which was completely abolished by the presence of PTP inhibitors. Taken together these data demonstrate that the presence of mutant huntingtin at physiologically relevant levels results in impaired Ca2+ handling by mitochondria which negatively impacts their function and hence likely impairs proper neuronal function.


Figure 7
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  		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 µM FCCP treatment 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 mitochondrial potential in response to 10 µM FCCP and similar results were obtained. Data are mean ± S.E. n = 3 separate experiments. C, wild type (black bars) and mutant (gray bars) cells were exposed to 1 nM 4-BrA23187 in the presence of increasing Ca2+ concentrations. Quantification of Mitotracker H2 ROS Red fluorescence, as relative units, shows significantly reduced mitochondrial potential in mutant cells pretreated with 4-BrA23187 (4-BrA) at each Ca2+ concentration 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) cells exposed to 1 nM 4-BrA23187 in the presence of increasing Ca2+ concentrations. Addition of 1 nM 4-BrA23187 plus different CaCl2 concentrations induced a significant 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).

 
Excitotoxicity has been suggested as a key mechanism that is responsible for neurodegeneration in HD (42), and dysfunction at the level of the mitochondria could be a mediator of this toxicity. Mitochondrial dysfunction could result in an increase in the sensitivity of neurons to glutamate, leading to Ca2+ induced cellular dysfunction and eventually cell death (the role of mitochondria in excitotoxicity is discussed in Refs. 43–45). It has been shown previously that decreases in oxidative phosphorylation and state 3 rates are early events in excitotoxicity, and occur prior to the commitment to cell death (46). Moreover, it has been shown that free µM Ca2+ concentrations cause significant decreases in oxidative phosphorylation in isolated mitochondria (29). To determine if mutant huntingtin alters mitochondrial response to Ca2+, we measured respiration rates in the presence of increasing Ca2+ concentrations in mitochondria from wild type (STHdhQ7/Q7) and mutant huntingtin (STHdhQ111/Q111)-expressing cells. We observed that the decrease in the state 3 rate (ADP phosphorylation rate) occurred at significantly lower Ca2+ concentrations in mutant cells compared with wild type cells, suggesting an increased sensitivity to Ca2+.

Elevated mitochondrial Ca2+ levels are usually associated with the opening of PTP. However, the mechanism of the oxidative phosphorylation decrease due to elevated Ca2+ levels is not quite clear (discussed in Ref. 46). It has been suggested that permeabilization of the outer mitochondrial membrane leads to activation of an alternative respiratory pathway, which utilizes exogenous NADH oxidation on the outer mitochondrial membrane and translocation of subsequently reduced cytochrome c to the inner membrane where it feeds into complex IV (34, 35). In our study, addition of NADH and cytochrome c caused an increase in the state 3 rates in the presence of Ca2+, but not in the absence, and cytochrome c alone did not increase state 3. These results indicate the Ca2+-induced decrease in oxidative phosphorylation is associated with increased permeabilization of the mitochondrial membrane but not with substantial loss of cytochrome c.

In this study respiration rates were measured in mitochondria isolated from STHdhQ7/Q7 (wild type) and STH-dhQ111/Q111 (mutant) cells in KCl-based EGTA containing respiration buffer, and no significant differences were observed. In our previous study, respiration rates were measured in digitonin-permeabilized cells, in a sucrose-based buffer without EGTA, and significant decreases in the state 3 rates were observed in STHdhQ111/Q111 (mutant) cells (18). In the light of the current findings, it is likely that the experimental conditions used in our previous study were permissive for the mutant huntingtin-dependent mitochondrial defects. These conditions resulted in the deficits observed in state 3 rates for the mutant cells. Indeed it is likely that the presence of free Ca2+ in the buffers (e.g. from the sucrose) likely resulted in an increase in mitochondrial membrane permeability to a greater extent in the mutant cells than the wild type cells, which caused the observed differences in respiration.

It has been shown that huntingtin associates with the outer mitochondrial membrane (47). We tested the integrity of outer mitochondrial membrane in basal conditions. We found that mitochondria from both cell lines have a good outer mitochondrial membrane integrity as state 3 rates did not increase in the presence of NADH and cytochrome c. The inner mitochondrial membrane was of good integrity as well, because NADH (inner mitochondrial membrane impermeable) did not work as respiratory substrate. Also, state 4 rates were comparable in the two cell lines, indicating similar levels of inner membrane proton leakage. However, it is still possible that changes in the integrity of mitochondrial membrane due to mutant huntingtin are quite subtle and could not be detected with the methods we used.

Mutant huntingtin-expressing cells (STHdhQ111/Q111) showed markedly enhanced {Delta}{Psi}m reduction in response to increasing Ca2+ concentrations both in vitro and in situ. Mitochondrial depolarization in response to Ca2+ is caused by Ca2+ uptake itself (partial and reversible depolarization) and by opening of the PTP when Ca2+ uptake capacity is exceeded (complete depolarization) (38). Because in our experiments {Delta}{Psi}m was measured in the population of mitochondria, determined {Delta}{Psi}m values could indicate the portion of mitochondria undergoing PTP associated with complete depolarization or {Delta}{Psi}m levels present in the majority of mitochondria at specific Ca2+ concentrations. As previously described, ADP prevents PTP opening and stabilizes {Delta}{Psi}m by the mechanism that includes binding and stabilization of adenine nucleotide translocator (ANT) in the conformation that prevents PTP opening (48). The differences in {Delta}{Psi}m reduction between wild type and mutant cells were significantly attenuated in the presence of ADP. This suggested that differences in the threshold for PTP opening, significantly contributed to {Delta}{Psi}m differences observed between wild type and mutant cells.

The reduction of {Delta}{Psi}m could be observed at as low as 0.4 and 0.6 µM Ca2+ (concentrations at which decreases in oxidative phosphorylation were observed) in the mutant but not in the wild type cells. Because respiration was first monitored in state 4 followed by the induction of state 3, the observed differences in the state 3 rates are likely caused by the differences in the {Delta}{Psi}m before the state 3 was induced. Indeed, no differences in {Delta}{Psi}m were observed at 0.4 and 0.6 µM Ca2+ when measured in state 3 conditions.

ROS production could contribute to Ca2+-induced PTP opening (discussed in Ref. 40). As described, determination of released H2O2 is a common and the most reliable measure of mitochondrial ROS production (39). When added to isolated mitochondria, Ca2+ caused a dose-dependent increase in the release of H2O2. However, no significant difference in released H2O2, between wild type and mutant, was observed at different Ca2+ concentrations (state 4). Therefore, the dramatic differences in {Delta}{Psi}m and Ca2+ uptake capacity observed between wild type and mutant are likely not caused by differences in the ROS production. However, in the presence of ADP (state 3), we observed higher levels of released H2O2 in mutant mitochondria, which reached significance at several data points. Increased ROS in the presence of ADP would likely contribute to more pronounced decrease in state 3 rates observed in mutant mitochondria in the presence of Ca2+. Unexpectedly, we observed reduced H2O2 release from mutant mitochondria upon rotenone treatment. Our results suggest that ROS production is likely not the mechanism for reduced threshold for PTP opening in the mutant cells. However, there was a trend for mitochondria from mutant cells to exhibit altered ROS homeostasis compared with wild type (modest increase in the presence of Ca2+ and ADP; decrease in the presence of rotenone).

In this article we observed that mitochondria from mutant cells had reduced Ca2+ uptake capacity compared with mitochondria from wild type cells. As suggested in multiple articles, decreases in mitochondrial Ca2+ uptake capacity could be caused by a decreased threshold for PTP opening (7, 49). In fact, when mitochondria from mutant cells were treated with PTP inhibitors (cyclosporine A, ADP, oligomycin), the defect in Ca2+ uptake was almost abolished. Decreased mitochondrial Ca2+ uptake has already been reported in other HD models. Panov et al. (7, 13) reported decreased Ca2+ uptake in mitochondria from HD lymphoblast cell line, and brain mitochondria from full-length huntingtin transgenic mice (YAC72). Recently, attenuated Ca2+ uptake was reported in muscle mitochondria from R6/2 mice (30). Interestingly, recombinant truncated mutant huntingtin resulted in significant mitochondrial swelling at lower Ca2+ loads than truncated wild type protein when added to isolated mouse liver mitochondria (47). This suggested that the mechanism of mutant huntingtin-induced mitochondrial dysfunction is possibly through its direct effects on mitochondria.

Using multiple mitochondrial functional assays, we demonstrated a mitochondrial Ca2+ handling defect in mutant (STHdhQ111/Q111) cells. Although Ca2+ overload could be the cause of the earlier PTP opening in the mutant mitochondria, our data suggest that it is the opening of the PTP at lower Ca2+ concentrations resulting in membrane depolarization rather than Ca2+ overload. ROS could contribute the PTP opening, but it did not correlate with the significant changes in {Delta}{Psi}m observed in the mutant cells, suggesting a different mechanism. Based on these and other findings, we hypothesize that the increased sensitivity of mutant mitochondria to increases in intracellular Ca2+ results in depolarization, and this is an upstream event in the pathological cascade followed by decreases in respiration and Ca2+ buffering capacity. In the light of these current findings, successful PTP inhibition would be beneficial, but, as already discussed in terms of excitotoxicity (38), limiting mitochondrial Ca2+ uptake by manipulating its setpoint (through Ca2+-uniporter inhibition or Na+-Ca2+-exchanger activation) is also likely to be beneficial in the treatment of HD.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grant NS041744. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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: gvwj{at}uab.edu.

2 The abbreviations used are: HD, Huntington disease; {Delta}{Psi}m, mitochondrial membrane potential; PTP, permeability transition pore; 3-NP, 3-nitropropionic acid; KRH, Krebs-Ringer-HEPES; CM-H2TMRos, MitoTracker® Red; JC-1,5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; RCR, respiratory control ratio; FCCP, carbonyl cyanide 4-(trifluoromethoxy)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. Back

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


   ACKNOWLEDGMENTS
 
We thank Dr. P. Bernardi for suggestions and advice regarding outer mitochondrial membrane integrity experiments.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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