Impaired Regulation of Brain Mitochondria by Extramitochondrial Ca2+ in Transgenic Huntington Disease Rats*

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt51Q), modeling the adult form of HD. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} up to 2 μm activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Ca}_{\mathrm{free}}^{2+}\) \end{document} above 2 μm inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not affect the Ca2+-dependent activation of respiration but reduced Ca2+-induced inhibition. Thus, Ca2+ activation was mediated exclusively by extramitochondrial Ca2+, whereas inhibition was promoted also by intramitochondrial Ca2+. In contrast, htt51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca2+ activation, and a higher susceptibility to Ca2+-dependent inhibition. Furthermore htt51Q mitochondria exhibited a diminished membrane potential stability in response to Ca2+, lower capacities and rates of Ca2+ accumulation, and a decreased Ca2+ threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca2+-induced inhibition of respiration of htt51Q mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca2+. In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca2+. We suggest that specific regulatory Ca2+ binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt51Q and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.

phorylation in HD 150Q striatal cells but only after N-methyl-Daspartate receptor-induced Ca 2ϩ stress (13). A Ca 2ϩ -induced decrease of respiration was also identified in mitochondria isolated from htt 111Q striatal cells (10). In addition, we could recognize a compromised mitochondrial function in fibroblasts from a HD patient with htt 43Q (14). Taken together, all these data imply a disturbed interaction between HD mitochondria and cytosolic Ca 2ϩ ([Ca 2ϩ ] cyt ), but the underlying targets and pathomechanisms have remained uncertain.
Elevated [Ca 2ϩ ] cyt , mitochondrial Ca 2ϩ uptake via the Ca 2ϩ uniporter, and subsequent intramitochondrial Ca 2ϩ accumulation (15) activate mitochondrial respiration due to stimulation of mitochondrial dehydrogenases (16). Elevated intramitochondrial Ca 2ϩ levels also induce permeability transition (PT) if distinct [Ca 2ϩ ] cyt thresholds are exceeded (17). In addition, a reduced mitochondrial membrane potential (⌬⌿) and a decreased redox pressure, defined by the ratio of NADH/ NAD ϩ , further reinforce the tendency for undergoing mitochondrial PT (17).
Recently activation of the mitochondrial malate/aspartate shuttle was observed at low, i.e. submicromolar, [Ca 2ϩ ] cyt . This effect was caused by a Ca 2ϩ -dependent stimulation of the glutamate/aspartate translocator (18,19). This carrier, termed aralar in brain mitochondria, was shown to also be involved in the transport of reducing hydrogen into mitochondria via a reconstituted malate/aspartate shuttle (18).
Considering this background, it was the first goal of this study to answer the question whether or not htt expQ is mitochondriotoxic and if so to identify the underlying pathomechanism. For this purpose, we investigated the mitochondrial function in a newly generated transgenic HD rat strain with 51 glutamine repeats (htt 51Q ) (20). In contrast to htt Q150 R6/2 mice, a model of the juvenile form of HD (21), the htt 51Q rat strain exhibits specifically an adult-related onset of the neurological HD phenotype (20).
Because HD-specific changes in mitochondrial function may be related to alterations in the intracellular Ca 2ϩ homeostasis (7-11, 22, 23), the second goal of this study was to evaluate the influence of extramitochondrial Ca 2ϩ on the function of wild type (WT) and htt 51Q mitochondria. We studied the effects of extramitochondrial Ca 2ϩ on mitochondrial Ca 2ϩ accumulation and oxidative phosphorylation in mitochondria isolated from striatum and brain. We found that extramitochondrial Ca 2ϩ in the submicromolar range was able to activate mitochondrial glutamate uptake, probably via binding on the high affinity Ca 2ϩ binding sites of the aspartate/glutamate translocator (aralar) that are exposed into the mitochondrial intermembrane space. Htt 51Q mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca 2ϩ activation, and a higher susceptibility to Ca 2ϩ -dependent inhibition. Furthermore htt 51Q mitochondria exhibited a diminished membrane potential stability in response to Ca 2ϩ , lower capacities and rates of Ca 2ϩ accumulation, and a decreased Ca 2ϩ threshold for PT in a substrate-independent but CsA-sensitive manner. Compared with WT, Ca 2ϩ -induced inhibition of respiration of htt 51Q mitochondria was less sensitive to ruthenium red (RR), indicating the involvement of extramitochondrial Ca 2ϩ . In summary, our data suggest that mitochondriotoxic actions of htt 51Q might be realized by affecting the regulatory Ca 2ϩ binding sites of mitochondrial carrier proteins like aralar and the PT pore, finally leading to energetic depression (6,24), mitochondrial cell death, and atrophy of affected tissues (6).

EXPERIMENTAL PROCEDURES
Animals-Transgenic animals were obtained from the central animal facility of the University of Tübingen, Tübingen, Germany. HD rats expressed 727 amino acids of the htt 51Q gene corresponding to 22% of the full-length gene. Tail tips were removed from all rats at the age of 3 weeks, and genotypes were determined by Southern blot analysis. We used male and female rats aged 21-27 months and compared them with agematched littermate WT rats from the same source. For some studies, normal adult Wistar WU rats were also used (Charles River Laboratories). All research and animal care procedures were performed according to European guidelines.
Isolation of Mitochondria-Mitochondria were prepared either from striatum or from the remaining part of the brain (total brain minus striatum) using a slightly modified protocol according to Kudin et al. (25), avoiding bovine serum albumin (BSA) during the preparation and investigation of mitochondria. In brief, animals were anesthetized with CO 2 and killed by decapitation. Brain tissue was immediately transferred into the ice-cold isolation medium (IM) consisting of 225 mM mannitol, 75 mM sucrose, 20 mM MOPS, 1 mM EGTA, and 0.5 mM dithiothreitol, pH 7.4. Then we minced the tissue, added 10 ml of nargase medium (IM ϩ 0.05% (w/v) nargase)/1 g of tissue, and homogenized the mixture with a glass/glass homogenizer. Then the homogenate was diluted 1:4 with nargase-free IM and centrifuged at 2,000 ϫ g for 4 min. After centrifugation, the supernatant was passed through a cheesecloth and centrifuged at 12,000 ϫ g for 9 min. To permeabilize the synaptosomes, the resulting pellet was suspended in 10 ml of ice-cold digitonin medium (IM ϩ 0.02% digitonin), transferred to a small glass homogenizer, and manually homogenized 8 -10 times to obtain a homogenous suspension. Finally the suspension was centrifuged at 12,000 ϫ g for 11 min, and the resulting pellet was suspended in 400 l of IM/g of tissue.
Respirometry-Mitochondrial respiration was measured with a Clark-type oxygen electrode by means of high resolution respirometry using an OROBOROS Oxygraph-2k (Oroboros, Innsbruck, Austria) (26) at 30°C. Respiration of mitochondria (0.06 mg of protein/ml) was investigated in EGTA medium (120 mM mannitol, 40 mM MOPS, 5 mM KH 2 PO 4 , 60 mM KCl, 5 mM MgCl 2 , and 0.1 mM EGTA, pH 7.4). Extramitochondrial concentrations of free Ca 2ϩ (Ca free 2ϩ ) were adjusted either by up to six sequential Ca 2ϩ additions (each of 20 M) or by one single addition of 50 M Ca 2ϩ into the medium. Ca free 2ϩ under each condition was verified by Fura-2 measurements as described below.
Measurement of Ca free 2ϩ in EGTA Medium-Ca free 2ϩ in the EGTA medium was measured fluorimetrically with Fura-2 (10 M) as described previously (28,29). Because the EGTA medium contained 5 mM Mg 2ϩ that competitively binds Fura-2 (29), the dissociation constant (K d ) of the Ca 2ϩ -Fura-2 complex was measured experimentally under these conditions 3 and found to be 0.3 M, which was similar to that found in a previous study (28).
⌬⌿ Measurements-Mitochondrial ⌬⌿ was monitored fluorimetrically by the release of safranine (30). Fluorescence was measured at 495 nm excitation and 586 nm emission using a Cary Eclipse fluorimeter (Varian) with 10 M safranine in stirred and thermostated cuvettes (30°C). Measurements were performed in medium A using mitochondria adjusted to 0.25 mg of protein/ml.
Protein Determination-Mitochondrial protein concentrations were determined by the bicinchoninic acid assay (31). BSA was used as standard.
Statistical Analysis-All results are presented as mean Ϯ S.D. or mean Ϯ S.E. as indicated. Statistical significance was analyzed by paired and unpaired two-tailed t test or Bonferroni's test.

RESULTS
Stimulation of Glutamate-dependent Respiration by Extramitochondrial Ca 2ϩ -To investigate the Ca 2ϩ dependence of oxidative phosphorylation, a model system allowing the stepwise increase of Ca free 2ϩ from nM to M concentration ranges was developed. We used an incubation medium containing 100 M EGTA to keep Ca free 2ϩ negligibly low (EGTA medium). Fig. 1A shows a respirogram of brain mitochondria isolated from normal rats using 10 mM glutamate and 2 mM malate as substrates. After addition of 2.5 mM ADP, an unusually low glutamate-dependent respiration rate (state 3 glu/ mal) was obtained. To clarify whether such a modest respiration resulted from an insufficient complex I-related metabolism, we measured the complex II-dependent respiration by applying the specific complex I inhibitor rotenone and succinate as complex II-specific substrate (state 3 suc respiration). As shown in Fig.  1A, succinate caused a normal state 3 suc respiration that clearly exceeded the state 3 glu/mal respiration. This finding suggests an inadequate complex I-mediated respiration. However, if the same experiment was performed in the presence of 1.35 M Ca free 2ϩ (Fig. 1B), the state 3 glu/mal respiration was more than doubled compared with that in Ca 2ϩ free conditions ( Fig. 1, A and B). Thus, the increase in Ca free 2ϩ induced a normalization of the mitochondrial complex I-dependent respiration in normal brain mitochondria.
In a second approach, state 3 glu/mal respiration was titrated by sequential Ca 2ϩ additions (Fig. 1C). Again state 3 glu/mal was very low under Ca 2ϩ free conditions but increased stepwisely until Ca free 2ϩ reached 2 M. At this Ca free 2ϩ concentration, the maximum state 3 glu/mal respiration was observed, whereas further Ca 2ϩ additions provoked an inhibitory effect. When Ca 2ϩ -induced respiration rates were normalized against the corresponding respiration rate under Ca 2ϩ -free conditions and plotted versus Ca free 2ϩ (Fig. 1F), about a 2-fold increase of state 3 glu/mal respiration by 2 M Ca free 2ϩ was revealed. To further ascertain whether the pronounced Ca 2ϩ stimulation of state 3 glu/mal respiration is a characteristic property of the glutamate metabolism, we investigated the substrate dependence of this effect in more detail. In particular, we considered pyruvate/malate as an alternative substrate of complex I-dependent respiration. We found that state 3 pyr/mal respiration was also significantly activated (16 Ϯ 3%) but to a much lesser extent than state 3 glu/mal (87 Ϯ 8%; Fig. 1, E and F). Additional experiments revealed that the activation of state 3 pyr/mal is not caused by extramitochondrial Ca 2ϩ but is a result of large time requirement for complete 3 C. Tanne and F. N. Gellerich, unpublished observation. activation of pyruvate dehydrogenase. State 3 suc respiration remainedunaffectedbyCa free 2ϩ (Fig.1D).Thus,glutamate/malatedependent respiration could be identified as the most sensitive target of Ca free 2ϩ . The Ca free 2ϩ concentration required for half-maximum activation of state 3 glu/mal respiration was 0.26 Ϯ 0.02 M. However, this value was much lower than the K m calculated for mitochondrial Ca 2ϩ accumulation via the Ca 2ϩ uniporter under similar conditions 4 (2.5 Ϯ 0.2 M). This finding suggests that Ca 2ϩ -dependent activation of state 3 glu/mal respiration cannot be mediated by the mitochondrial Ca 2ϩ uniporter. If this assumption is valid, state 3 glu/mal respiration should also be activated by Ca free 2ϩ in the presence of RR, an inhibitor of the Ca 2ϩ uniporter. Indeed 250 nM RR, a dose able to block mitochondrial Ca 2ϩ uptake through the Ca 2ϩ uniporter completely under the conditions used here (not shown), did not prevent the Ca free 2ϩ -dependent state 3 glu/mal activation (Fig. 1F). Thus, mechanisms underlying the stimulation of glutamate/malatedependent respiration do not require intramitochondrial Ca 2ϩ accumulation and therefore must be initiated outside the Ca 2ϩimpermeable mitochondrial inner membrane.
Glutamate is taken up by mitochondria either via aralar, leading to its subsequent transamination by aspartate aminotransferase (inhibitable by aminooxyacetate (AOA)), or via the glutamate/OH carrier followed by its desamination by glutamate dehydrogenase (19,(32)(33)(34). However, the activity of the glutamate/OH carrier is low in most organs except liver and kidney (34). If so, Ca 2ϩ activation of state 3 glu/mal respiration should be inhibited by AOA. Indeed the Ca 2ϩ -dependent state 3 glu/mal activation was significantly suppressed by 2 mM AOA 5 (Ϫ55 Ϯ 5%), confirming the involvement of the aralar/transaminase pathway in the Ca 2ϩ activation. In addition, we found that AOA (250 nM) did not affect the mitochondrial respiration with pyru-vate/malate as substrates 5 (not shown). It is therefore likely that activation of state 3 glu/mal respiration by extramitochondrial Ca free 2ϩ is mediated by an enhanced glutamate transport into the mitochondrial matrix via aralar.
However, the functional relationship between Ca free 2ϩ and state 3 glu/mal respiration rate exhibited a biphasic nature. At 6.3 M or higher Ca free 2ϩ , a marked decrease in state 3 glu/mal respiration was monitored ( Fig. 1, C and F). Because this change was significantly attenuated by 1 M CsA (data not shown), it was most likely caused by partial opening of the PT pore due to an intramitochondrial Ca 2ϩ overload. This conclusion was further validated by the following finding. In contrast to the Ca 2ϩ -dependent activation of state 3 glu/mal respiration, Ca 2ϩ -dependent inhibition of state 3 glu/mal respiration at high Ca free 2ϩ was diminished by 2 M RR (Fig. 1F). We therefore suppose that an intramitochondrial Ca 2ϩ overload via the Ca 2ϩ uniporter induces PT in a CsA-sensitive and thus cyclophilin D-dependent manner. Accordingly a significant inhibition of respiration by elevated Ca free 2ϩ was also found with pyruvate/malate and to a lesser extent with succinate ( Fig. 1, D, E, and F). Impaired Oxidative Phosphorylation of Brain Mitochondria from htt 51Q Rats-In the next series of experiments, htt 51Q and WT brain mitochondria were compared in EGTA medium in the presence of various substrates. State 3 respiration was adjusted by 2.5 mM ADP, whereas Ca free 2ϩ was kept low initially. As shown in Table 1, state 3 glu/mal (Ϫ31%) and state 3 pyr/mal (Ϫ32%) respiration of htt 51Q mitochondria was lowered significantly compared with WT. Concomitant reductions (between Ϫ20 and Ϫ29%) were also observed in the respiratory control indices (RCIs; calculated as the ratio of state 3/state 2 respiration) with NADH-linked complex I substrates. In contrast, complex II-dependent state 3 suc respiration of htt 51Q mito-

Impaired mitochondrial function in isolated brain mitochondria of HD rats
The mitochondria from 24-month-old htt 51Q and 18 -27-month-old WT rats were investigated in EGTA medium with different substrates (10 mM glutamate plus 2 mM malate, 10 mM pyruvate plus 2 mM malate, and 10 mM succinate and 1.6 M rotenone) without and with 250 nM RR as shown in Fig. 2. Non-Ca 2ϩ -stimulated mitochondrial respiration was measured in the presence of 2.5 mM ADP but without Ca 2ϩ addition. The extent of Ca 2ϩ stimulation was calculated as the difference between state 3 respiration rate measured at Ca free 2ϩ (either at 0.9 or 2.0 M) causing the maximum respiration and state 3 rate measured prior to Ca 2ϩ addition. The extent of Ca 2ϩ inhibition was calculated as the difference between the maximum Ca 2ϩ -stimulated state 3 rate and state 3 rate at maximum Ca free 2ϩ (32 M). The respiration rates are expressed in nmol of O 2 /min/mg of mitochondrial protein. The RCI values were calculated as the ratio of state 3 measured without Ca 2ϩ and state 2 respiration obtained before the addition of ADP. Data are given as mean Ϯ S.E. The number of measurements is indicated in parentheses.

Condition
WT chondria was only 12-15% decreased compared with WT, and the RCI suc remained unchanged ( Fig. 2H and Table 1). Typical Ca 2ϩ titration respirograms of WT and htt 51Q brain mitochondria with glutamate/malate as substrates are shown in Fig. 2, A-D. As demonstrated for normal mitochondria (Fig.  1C), state 3 glu/mal respiration of WT mitochondria was stimulated by three sequential Ca 2ϩ additions (Fig. 2, A and E), revealing a maximum respiration rate at 2 M Ca free 2ϩ . Further Ca 2ϩ additions caused a progressive inhibition of state 3 glu/mal respiration probably because of increasing mitochondrial Ca 2ϩ accumulation. RR prevented this and shifted the maximum state 3 glu/mal respiration from 2.0 to 6.3 M Ca free 2ϩ (Fig. 2, B and E). Similar results were obtained when pyruvate/malate were used as substrates ( Fig. 2G and Table 1). Compared with WT, htt 51Q mitochondria were characterized by a lower basal state 3 respiration prior to the first Ca 2ϩ addition regardless of whether glutamate/malate or pyruvate/malate were used (Fig.  2, E and G, and Table 1). However, maximum state 3 glu/mal respiration of htt 51Q mitochondria was already reached at 0.9 M Ca free 2ϩ , instead of 2 M Ca 2ϩ determined in WT mitochondria, but was much lower than the corresponding respiration of WT mitochondria (Fig. 2E). Contrarily the Ca 2ϩ -dependent peak of state 3 pyr/mal of htt 51Q mitochondria remained at 0.9 M Ca free 2ϩ and thus unchanged compared with WT (Fig. 2G). Furthermore the inhibition of state 3 respiration by higher Ca free 2ϩ levels was more pronounced in htt 51Q than in WT mitochondria, an effect that was again substrate-independent (Fig. 2, A, C, and E-H, and Table 1).
In general, RR did not influence the Ca 2ϩ -dependent activation of state 3 glu/mal and state 3 pyr/mal respiration (Fig. 2, B, D, F, and G, and Table 1). However, we could identify significant RR effects on the Ca 2ϩ -induced inhibition of state 3 respiration regardless of whether htt 51Q or WT mitochondria were studied and independently of the substrates used (Fig. 2, B, D, F, G, and H, and Table 1). Interestingly the extent of RR effects on state 3 respiration was larger in WT mitochondria (Fig. 2, F and G, and Table  1), indicating that extramitochondrial Ca free 2ϩ increased the tendency for PT pore opening in htt 51Q mitochondria. Furthermore RR shifted the maximum state 3 respiration rates with glutamate/malate or pyruvate/malate to higher Ca free 2ϩ levels exclusively in WT mitochondria (Fig. 2, E-G). In contrast, RRdependent maximum state 3 rates of htt 51Q mitochondria were moved toward lower Ca free 2ϩ levels as for pyruvate/malate or remained unchanged as for glutamate/malate (Fig. 2, E-G). We also addressed the issue of whether complex II-specific state 3 respiration, realized by the simultaneous application of the FADH-generating substrate succinate and the specific complex I inhibitor rotenone, would be affected by Ca free 2ϩ , RR, and/or htt 51Q . Under Ca 2ϩ -free conditions, the state 3 suc respiration of WT and htt 51Q mitochondria was comparable to or even higher than the corresponding complex I-linked respiration with glutamate/malate or pyruvate/malate (Figs. 1D and 2H and Table 1). Furthermore Ca free 2ϩ did not exert any activation of state 3 suc respiration either in WT or in htt 51Q mitochondria. Nevertheless there was a moderate and Ca 2ϩdependent inhibition of state 3 suc respiration if Ca free 2ϩ was increased into the range of 6.3-32 M (Figs. 1D and 2H). Although slightly more pronounced in htt 51Q mitochondria than in WT, this effect was in general smaller compared with complex I substrates (Table 1). In line with the results obtained with complex I-specific substrates, RR did not alter the basal state 3 suc respiration under Ca 2ϩ -free conditions but reduced significantly the Ca 2ϩ -dependent inhibition of respiration at high Ca free 2ϩ levels. Notably this effect was exclusively observed in WT mitochondria but not in htt 51Q mitochondria ( Fig. 2H and Table 1).
For a more detailed evaluation of substrate dependences of htt 51Q -specific impairments of mitochondrial function, relative respiration deficiencies were calculated as the decrease of state 3 respiration in relation to corresponding WT values (Fig. 2,  E-H). In general, these calculations were based on experimental data obtained in the absence of RR. For both complex I-de- pendent substrates used here, glutamate/malate and pyruvate/ malate, the relative respiration deficiency was about 25% under Ca free 2ϩ conditions but increased continuously to about 60% at maximum Ca free 2ϩ levels (32 M; Fig. 2, E and G). In contrast, the relative respiration deficiency was only 12% prior to the first Ca 2ϩ addition and increased to 34% at 32 M Ca free 2ϩ if succinate was used as complex II-specific substrate (Fig. 2H).
In the next step, we addressed the question of which parts of oxidative phosphorylation, i.e. ⌬ generation (respiration) and/or ⌬ utilization (ADP phosphorylation and Ca 2ϩ accumulation), contribute to htt 51Q -specific changes of brain mitochondria. Respiration and ADP phosphorylation were experimentally separated by the uncoupler FCCP, which was added to mitochondria after the induction of state 3 respiration by 100 M ADP. However, state 3 glu/mal respiration was not affected by FCCP either in htt 51Q or in WT brain mitochondria (not shown). Similar results were obtained with pyruvate/malate and succinate as substrates (not shown). We therefore concluded that ADP phosphorylation was not compromised in htt 51Q mitochondria. Rather htt 51Q -dependent changes of mitochondrial function might be caused by impairments within the ⌬⌿ generating metabolism and/or by altered PT.
Decreased Stability of the Inner Membrane Potential ⌬⌿ of htt 51Q Mitochondria-To study the underlying mechanism(s) of htt 51Q -dependent changes in the ⌬⌿ generating metabolism of oxidative phosphorylation, we asked whether htt 51Q is able to affect the stability of ⌬⌿. Therefore, the influence of Ca free 2ϩ on ⌬⌿ was investigated in EGTA-free medium using the ⌬⌿-sensitive fluorescence indicator safranine and different substrates (30). As shown in Fig. 3, A and B, glutamate/malate-consuming WT and corresponding htt 51Q brain mitochondria accumulated safranine to a similar extent, indicating comparable membrane potentials in both types of mitochondria. After the first addition of 10 M Ca 2ϩ and subsequent mitochondrial Ca 2ϩ uptake, WT brain mitochondria released 42 Ϯ 15% of their total safranine (Fig. 3, A and C). The corresponding fluorescence increase visualized a Ca 2ϩ -dependent decrease of ⌬⌿. The following Ca 2ϩ additions (each 10 M Ca 2ϩ ) induced a further but stepwisely reduced depolarization. Finally FCCP (70 nM) was applied to abolish the residual electrochemical proton gradient of the inner mitochondrial membrane and thus to abrogate ⌬⌿. In contrast to WT mitochondria, htt 51Q mitochondria released 72 Ϯ 14% of their total safranine already after the first Ca 2ϩ addition (Fig. 3, B and C). Similar results were obtained with pyruvate/malate or succinate as complex I-and complex IIspecific substrates, respectively (not shown). These findings revealed a significantly higher sensitivity of ⌬⌿ of htt 51Q mitochondria against destabilizing effects of extramitochondrial Ca 2ϩ than WT mitochondria. Impaired Ca 2ϩ Accumulation of htt 51Q Mitochondria-Because the mitochondrial Ca 2ϩ uniporter is driven by ⌬⌿ of the inner mitochondrial membrane, the htt 51Q -promoted Ca 2ϩ sensitivity of ⌬⌿ may have an impact on the kinetics of mitochondrial Ca 2ϩ uptake and accumulation. To prove this assumption, isolated brain mitochondria were kept in medium A containing the fluorescence indicator Calcium Green to monitor extramitochondrial Ca free 2ϩ and either 10 mM glutamate and 2 mM malate, 10 mM pyruvate and 2 mM malate, or 10 mM succinate and 2 M rotenone (Figs. 4 and 5). Mitochondrial Ca 2ϩ accumulation rates were calculated as the first derivative of fluorescence intensity versus time. Ca 2ϩ added with the first addition (10 M) was taken up by htt 51Q mitochondria to a large   Fig. 4A, this process was reflected by a fast fluorescence increase due to the binding of added Ca 2ϩ to extramitochondrial Calcium Green followed by a slower fluorescence decrease almost to levels seen before Ca 2ϩ addition. The latter fluorescence decrease displayed mitochondrial Ca 2ϩ accumulation. In contrast, the second Ca 2ϩ addition (10 M) was only partially accumulated by htt 51Q mitochondria (25% instead of 100%). Already after the third Ca 2ϩ addition, htt 51Q mitochondria started to release Ca 2ϩ into the medium possibly via PT pore opening. This scenario remained unchanged if pyruvate/malate (Fig. 5E) or succinate/rotenone (Fig. 5G) instead of glutamate/malate were used. However, when htt 51Q mitochondria were compared with WT mitochondria, two major differences became evident. (i) In response to sequential Ca 2ϩ additions (each 10 M), Ca 2ϩ was completely accumulated by WT mitochondria until the fifth addition (Fig. 4B), meaning that WT mitochondria are characterized by a significantly higher Ca 2ϩ accumulation capacity than htt 51Q mitochondria. (ii) The velocity of mitochondrial Ca 2ϩ uptake, illustrated by the slope of the decline of repetitive Calcium Green spikes (Fig.  4, A-D, lower traces) was significantly higher in WT than in htt 51Q mitochondria.

extent. As seen in
A further significant alteration was identified by comparing mitochondria from brain and striatum (Fig. 4). Striatal WT mitochondria revealed about a 3 times lower Ca 2ϩ accumulation rate than those from the rest of the brain (Fig. 4,  compare B and D, lower traces). On the other hand, Ca 2ϩ accumulation rates were similarly reduced in htt 51Q mitochondria regardless of whether they were derived from brain or striatum (Fig. 4, compare A and C, lower traces). We also analyzed mitochondrial Ca 2ϩ accumulation indices that were calculated as the product of Ca 2ϩ accumulation rate and corresponding completeness of mitochondrial Ca 2ϩ uptake. The completeness of mitochondrial Ca 2ϩ uptake was defined as the ratio of remaining Calcium Green signal after mitochondrial Ca 2ϩ uptake (Fig. 4A, b)/maximum Calcium Green signal after the previous Ca 2ϩ addition (Fig. 4A, a). As illustrated in Fig. 4E, Ca 2ϩ accumulation indices were signif-icantly different between htt 51Q and WT mitochondria. Furthermore Ca 2ϩ thresholds linked to PT pore opening and mitochondrial Ca 2ϩ efflux (Fig. 4, A-D) were markedly decreased in htt 51Q mitochondria compared with WT (Fig.  4F). Reduced Ca 2ϩ thresholds and Ca 2ϩ accumulation rates of htt 51Q mitochondria were also observed with pyruvate/ malate (Fig. 5, E and F) and with succinate/rotenone (Fig. 5G).
CsA Normalizes the Impaired Ca 2ϩ Accumulation of htt 51Q Brain Mitochondria in Vitro-If Ca 2ϩ -induced functional impairments of htt 51Q mitochondria (Fig. 4) were related to the opening of the PT pore, CsA should diminish this effect. To verify this, the influence of CsA (2 M) on the kinetics of mitochondrial Ca 2ϩ accumulation was monitored with Calcium Green (Fig. 5). The inhibiting effect of CsA on the PT in isolated brain mitochondria is known to depend on the composition of the incubation medium (35)(36)(37), i.e. for maximum effects of CsA, the presence of ADP and Mg 2ϩ is required. 6 Thus, CsA effects on htt 51Q mitochondria were studied in Mg 2ϩ and ADP-containing medium B. Again htt 51Q mitochondria showed their typical features as a low Ca 2ϩ uptake rate, a reduced capacity of Ca 2ϩ accumulation, and an early progression to PT (Fig. 5, A, E, and G). However, all these changes were diminished by 2 M CsA regardless of whether glutamate/malate, pyruvate/malate, or succinate/ rotenone were consumed (Fig. 5, A, B, E, F, G, and H). Consequently Ca 2ϩ accumulation curves became like those of WT mitochondria (Fig. 5C). Furthermore CsA caused a significant increase in the Ca 2ϩ thresholds linked to PT pore opening and mitochondrial Ca 2ϩ release but to an extent similar to those in htt 51Q and WT mitochondria (not shown). As illustrated in Fig. 5H, CsA increased the Ca 2ϩ accumulation indices (see definition above) following the first three Ca 2ϩ additions to htt 51Q mitochondria by about 96 -158%. In line with this finding, there was a only a low CsA effect (18 Ϯ 12%) on Ca 2ϩ accumulation rates of WT mitochondria 6 Z. Gizatullina and F. N. Gellerich, unpublished observation.  CsA (B, D, F, and H). Calcium Green was used to monitor extramitochondrial Ca 2ϩ levels in response to sequential Ca total 2ϩ additions (10 M each). Calcium Green fluorescence (upper recordings) in arbitrary units (A.U.) and its first derivative, corresponding to the rate of mitochondrial Ca 2ϩ uptake (lower recordings), are presented. H, htt 51Q -dependent Ca 2ϩ accumulation indices (see Fig. 3 for explanation) in the presence of 2 M CsA were normalized against the corresponding values obtained without CsA using the substrates glutamate/malate (GM; n ϭ 5), pyruvate/malate (PM; n ϭ 7), and succinate/rotenone (SR; n ϭ 4). *, p Ͻ 0.05 compared with CsA-free conditions. exposed to glutamate/malate (Fig. 5, C and D), pyruvate/ malate, or succinate rotenone (not shown).

Activation of Oxidative Phosphorylation by Extramitochondrial Ca 2ϩ : a Novel
Mechanism of Regulation-For the first time, this study reveals that state 3 glu/mal respiration of brain mitochondria is strongly and exclusively activated by extramitochondrial Ca 2ϩ in the nanomolar to low micromolar concentration range. This finding might reflect an important mechanism for the regulation of oxidative phosphorylation by Ca 2ϩ . To explain this type of activation, the presence of specific regulatory Ca 2ϩ binding sites as metabolic sensors of extramitochondrial Ca 2ϩ in the outer compartment of mitochondria must be considered. Aralar, the cerebral isoform of the Ca 2ϩdependent aspartate/glutamate carrier, serves as an important component of the mitochondrial malate/aspartate shuttle. As recently reported, aralar contains a Ca 2ϩ binding site that is localized in the intermembrane space and regulates the activity of the aspartate/glutamate carrier in a Ca 2ϩ -dependent manner (18,19). Two basic findings of our study support an involvement of aralar in the regulation of oxidative phosphorylation by extramitochondrial Ca 2ϩ . (i) This carrier is activated by low Ca 2ϩ concentrations (S 0.5 ϭ 0.3 M Ca free 2ϩ ) (18). Such Ca 2ϩ levels are very close to those required for the Ca 2ϩ activation of state 3 glu/mal identified under our experimental conditions. (ii) Ca 2ϩ -stimulated state 3 glu/mal respiration was suppressed by AOA, an inhibitor of the mitochondrial aspartate aminotransferase, which metabolizes glutamate after its transport into the mitochondrial matrix via aralar. Consistently Ca 2ϩ -stimulated state 3 glu/mal respiration was not affected by RR, an inhibitor of mitochondrial uptake by the Ca 2ϩ uniporter.
In line with our results, Pardo et al. (18) have demonstrated an activation of the malate/aspartate shuttle by Ca 2ϩ via an increased transport of reducing hydrogen into brain mitochondria. Furthermore Palmieri et al. (19) could detect a Ca 2ϩ -activation of the glutamate/aspartate carrier because of increased rates of mitochondrial glutamate decarboxylation in the human cell line HEK-293T. An example for the importance of aralar has been also shown in patients with Mohr-Tranebjaerg syndrome, which is characterized by a decreased NADH shuttle activity due to mutations in the TIMM8a protein complex. TIMM8a is responsible for the import of the aspartate/glutamate carrier protein into mitochondria (38).
In contrast to the Ca 2ϩ -dependent activation of state 3 glu/mal and state 3 pyr/mal respiration by nanomolar and low micromolar Ca free 2ϩ levels, mitochondrial respiration was rather inhibited if Ca free 2ϩ was increased to Ͼ2.0 M (Fig. 1). Because of its pronounced sensitivity to RR, this Ca 2ϩ -dependent inhibition of state 3 respiration was most likely caused by the intramitochondrial accumulation of Ca 2ϩ following its uptake via the Ca 2ϩ uniporter. Because mitochondrial inhibition was also attenuated by CsA, it became evident that PT pore opening, induced by intramitochondrial Ca 2ϩ accumulation, must be involved in the suppression of respiration.
Association of HD with Impaired Regulation of Oxidative Phosphorylation by Extramitochondrial Ca 2ϩ -This study identified a deficient oxidative phosphorylation in the brain of htt 51Q rats ( Fig. 2 and Table 1). Compared with WT, mitochondria of htt 51Q donors were characterized by a 30 -34% lower state 3 respiration prior to extramitochondrial Ca 2ϩ additions regardless of whether complex I-or complex II-dependent substrates were used (Table 1 and Fig. 2, E, G, and H). In general, htt 51Q mitochondria were less prone to activation but more susceptible to inhibition by Ca 2ϩ and unable to keep ⌬⌿ stable during the Ca 2ϩ influx. The state 3 glu/mal of WT mitochondria was stable against elevations of Ca free 2ϩ concentrations up to 2 M, whereas the htt 51Q mitochondria were stable up to 0.9 M Ca free 2ϩ only. In contrast, the maximum state 3 pyr/mal at 0.9 M Ca free 2ϩ remained unaffected by htt 51Q . This finding reflects a stronger effect of extramitochondrial Ca 2ϩ on the glutamate/ malate respiration than on pyruvate/malate respiration of htt 51Q mitochondria.
Because the uncoupled respiration (induced by FCCP) was similar to the state 3 respiration of htt 51Q and WT mitochondria, a defect of ADP phosphorylation itself can be excluded to underlie mitochondriotoxic effects of htt 51Q (39). Thus, not the system utilizing the proton motive force but rather the machinery generating this force must be deficient in htt 51Q mitochondria. This kind of insufficiency was clearly substrate-specific because the complex I-dependent respiration of glutamate/ malate or pyruvate/malate was more affected than the complex II-specific consumption of succinate ( Fig. 2 and Table 1). This finding fits well with our recent study revealing a decreased state 3 I/II ratio of skeletal muscle mitochondria from HD R6/2 mice compared with WT animals (8).
The larger relative deficiency of HD mitochondria utilizing complex I-dependent substrates (Fig. 2, E-H) could be caused by the following mechanism. At low Ca free 2ϩ , activation of state 3 respiration by extramitochondrial Ca 2ϩ might be hindered because of interactions of htt 51Q with regulatory Ca 2ϩ binding sites of mitochondrial substrate carriers such as aralar. At high Ca free 2ϩ levels and intramitochondrial Ca 2ϩ accumulation, inhibition of complex I-dependent respiration may be at least partially caused by the release of NADH and NAD ϩ from the matrix space due to PT pore opening. The latter effect was seen by an increase of respiration after NADH addition to brain mitochondria exposed to inhibiting Ca 2ϩ levels (not shown). A similar effect of NADH was demonstrated recently in HD mitochondria from striatal neurons (10). In contrast, under the same conditions of high Ca free 2ϩ , the succinate respiration was not dramatically affected likely because the coenzyme FAD is covalently bound to the succinate dehydrogenase and therefore cannot leave the mitochondrial matrix during PT. Direct measurements of PT of htt 51Q mitochondria with Calcium Green revealed significantly reduced Ca 2ϩ thresholds and decreased Ca 2ϩ accumulation rates for complex I-and complex II-dependent substrates (Figs. 4 and 5). Previously we identified decreased Ca 2ϩ thresholds for PT in skeletal muscle mitochondria of htt 140Q R6/2 mice with both succinate/rotenone and pyruvate/malate as substrates (8). These data implicate an impaired regulation of mitochondrial PT possibly because of an interaction of htt 51Q with the PT pore.
HD-specific impairments of the Ca 2ϩ -dependent regulation of mitochondrial function are in line with several previous reports (7)(8)(9)(10)(11)14). For example, instability of ⌬⌿ and lower Ca 2ϩ accumulation capacities of HD mitochondria have been identified in lymphocytes of HD patients (7); in brain, liver, and muscle of transgenic mice (7)(8)(9); and in spiny striatal neurons containing htt exp (10). Swelling of liver mitochondria of htt 111Q knock-in mice was shown to be more sensitive to Ca 2ϩ additions compared with WT mitochondria (9). However, consensus in this issue is lacking because other investigators have either not observed an increased Ca 2ϩ sensitivity of PT (12) or observed an enlarged Ca 2ϩ loading capacity (13) of mitochondria in HD mice models. Most probably, methodological differences underlie these conflicting results. For instance, studies by Brustovetsky et al. (12) and Oliveira et al. (13) were performed using non-synaptosomal mitochondria isolated and assessed in the presence of BSA. Notably Panov et al. (40) found that the defective Ca 2ϩ handling of brain mitochondria can only be observed consistently if these organelles are isolated in the absence of BSA. Presumably BSA displaces htt expQ from its binding places in mitochondria. Therefore, we generally avoided the exposure of mitochondria to BSA during isolation and experiments.
A further difference compared with previous studies concerns the isolation of brain mitochondria. Brustovetsky et al. (12) and Oliveira et al. (13) used mitochondria purified with a Percoll gradient that systematically excludes synaptosomal mitochondria. In contrast, we utilized non-synaptosomal and synaptosomal mitochondria according to the method described by Kunz and coworkers (25). This aspect is important because it considers the greater susceptibility of synaptosomal mitochondria to PT because of their higher content of cyclophilin D compared with non-synaptosomal mitochondria (41). Thus, isolated non-synaptosomal HD mitochondria could be more resistant to Ca 2ϩ challenges than a more physiological mixture of both fractions.
Up to now, the nature of htt expQ -caused cytotoxicity is unclear. Because binding of htt and htt expQ at the outer mitochondrial membrane has been detected (7,9,11,42), specific interactions with mitochondrial target proteins should be taken into account. Aralar may represent one of the target candidates that is affected by htt 51Q via its regulatory Ca 2ϩ binding site. However, other proteins exposing their regulatory binding sites to extramitochondrial Ca 2ϩ probably also play a role in htt 51Qinduced dysregulation of mitochondria. For example, Bernardi et al. (43) have described an external Ca 2ϩ binding site of the PT pore. Based on this, it has been postulated that cytosolic Ca 2ϩ decreases the opening probability of the pore by interfering with this site (43). In contrast, Kowaltowski and Castilho (44) postulate a stimulation of PT by extramitochondrial Ca 2ϩ . Our finding that the inhibition of mitochondrial Ca 2ϩ uptake by RR diminished the inhibition of state 3 respiration by Ca 2ϩ levels above 2.0 M largely in WT but only to a minor extent in htt 51Q mitochondria ( Fig. 2 and Table 1) supports the hypothesis of Bernardi et al. (43). Indeed we provide the first evidence for an interaction of htt 51Q with a regulatory Ca 2ϩ binding site of the PT pore by showing that htt 51Q effects are opposite on aralar and PT. Aralar is insufficiently activated followed by a limited mitochondrial substrate supply and thus a decreased activation of respiration by extramitochondrial Ca 2ϩ concentrations Յ2 M. In contrast, htt 51Q sensitizes the PT pore to extramitochondrial Ca 2ϩ levels Ͼ1 M, leading to pore opening and decreased respiration rates even in the presence of RR. It is therefore conceivable that htt 51Q interacts with the Ca 2ϩ binding site of the PT pore, thereby blocking a protective effect of extramitochondrial Ca 2ϩ against PT. This effect could explain the increased susceptibility of htt 51Q mitochondria to PT and their compromised Ca 2ϩ retention capacity.
Besides aralar and the PT pore, further potential target proteins of htt expQ deserve attention, e.g. a subfamily of Ca 2ϩ -binding mitochondrial carriers (45)(46)(47). These carriers are isoenzymes of the ATP-Mg/P i transporter responsible for the net influx or efflux of adenine nucleotides into or from mitochondria (48). Their Ca 2ϩ binding moieties in the N terminus may serve as sensors of cytosolic Ca 2ϩ . Notably the mitochondrial Ca 2ϩ uniporter itself provides a regulatory Ca 2ϩ binding site at the outer side of mitochondrial inner membrane (15,49). Interactions of htt expQ with this site could decrease the Ca 2ϩ accumulation of HD mitochondria. Finally the porin pore of the mitochondrial outer membrane, termed the voltage-dependent anion channel (VDAC), may also be regulated by cytosolic Ca 2ϩ (50). Htt 51Q -dependent changes of mitochondrial regulation may uncover important pathophysiological consequences. For instance, decreased Ca 2ϩ thresholds of htt expQ mitochondria for undergoing PT might be responsible for accelerated mitochondrial cell death, in particular under conditions of elevated cytosolic Ca 2ϩ concentrations seen in HD (6). Moreover impairments of HD mitochondria may limit the cellular function also at physiological Ca 2ϩ levels.
The protective CsA effects on the kinetics of Ca 2ϩ accumulation might contribute to a normalization of the Ca 2ϩ homeostasis in affected HD tissues. These assumption fits with recent studies showing that CsA is able to increase and normalize the mitochondrial Ca 2ϩ uptake threshold of HD mitochondria from clonal striatal cells (10).
As a novel finding, we demonstrated that protective CsA effects occur not only under conditions of Ca 2ϩ overload but also within physiological concentration ranges of extramitochondrial Ca 2ϩ in htt 51Q mitochondria. This may have important, disease-specific consequences in vivo when an altered Ca 2ϩ homeostasis affects the energy metabolism and vice versa.
It is widely accepted that HD attacks preferably the striatum, whereas other brain regions are less affected. We have addressed this issue by analyzing the function of mitochondria from striatum and the remaining brain regions within most of the protocols used. We found that the mechanisms by which htt 51Q modifies mitochondrial function might not differ between these two sources of mitochondria.
In summary, our data provide several lines of evidence for htt 51Q -induced pathomechanisms underlying the initiation and progression of HD. Ca 2ϩ -dependent impairments of mitochondrial oxidative phosphorylation due to a limited substrate supply and/or altered PT are suggested as major causes for the increased vulnerability of HD mitochondria against Ca 2ϩ stress. The protective effect of CsA may provide a basis for the development of new therapeutic modalities.