Archives of Biochemistry and Biophysics 579 (2015) 40–46

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Knockout of cyclophilin D in Ppif mitochondria against Ca2+ stress

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mice increases stability of brain

T. Gainutdinov a,b, J.D. Molkentin c, D. Siemen a, M. Ziemer a, G. Debska-Vielhaber a, S. Vielhaber a, Z. Gizatullina d, Z. Orynbayeva e,⇑, F.N. Gellerich a,d,⇑ a

Department of Neurology, Otto-von-Guericke-University, Magdeburg D-39120, Germany Institute of Ecology and Use of Mineral Resources, Academy of Sciences of Tatarstan, Kazan 420087, Russian Federation Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Howard Hughes Medical Institute, Cincinnati, OH 45229, USA d Leibniz Institute for Neurobiology, Brennecke Str. 6, Magdeburg D-39118, Germany e Department of Surgery, Drexel University College of Medicine, Philadelphia, PA 19102, USA b c

a r t i c l e

i n f o

Article history: Received 18 January 2015 and in revised form 24 May 2015 Available online 29 May 2015 Keywords: Cyclophilin D Cyclosporin A Brain mitochondria Glutamate Complex I respiration Mitochondrial permeability transition Ca2+ stress

a b s t r a c t The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BMWT) and CypD knockout (Ppif / ) mice (BMKO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca2+ stress conditions. Our data demonstrate that BMKO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca2+ effects than BMWT. Under the elevated Ca2+ and correspondingly decreased membrane potential the dose response in BMKO shifts to higher Ca2+ concentrations as compared to BMWT. However, significantly high Ca2+ levels result in complete loss of membrane potential in BMKO, too. CsA diminishes the loss of membrane potential in BMWT but has no protecting effect in BMKO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca2+. Due to missing of CypD the BMKO can favor PT only at high Ca2+ concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca2+ stress. Ó 2015 Published by Elsevier Inc.

Introduction Cyclophilin D (CypD1) is a member of the family of peptidyl-prolyl cis–transisomerases, which is known to facilitate the mitochondrial permeability transition pore (PTP) assembly [1]. Primarily using mouse models of genetically ablated CypD (Ppif / ) it has been shown that CypD plays an important role in the development of certain pathological conditions, such as ischemic heart [2] and brain injury [3], neurodegenerative diseases, including

⇑ Corresponding authors at: Department of Behavioral Neurology, Leibniz Institute for Neurobiology, Brennecke Str. 6, Magdeburg D-39118, Germany. Fax: +49 391 6715216 (F.N. Gellerich). Department of Surgery, Drexel University College of Medicine, 245 N 15th Street, Philadelphia, PA 19102, USA. Fax: +1 215 762 8389 (Z. Orynbayeva). E-mail addresses: [email protected] (F.N. Gellerich), zorynbay@ drexelmed.edu (Z. Orynbayeva). 1 Abbreviations: CypD, Cyclophilin D; CsA, cyclosporin A; RCI, Respiratory control indices; BM, brain mitochondria; MMSE, medium containing mannitol, MOPS, sucrose, and EGTA; MMMPK, medium containing MgCl2, mannitol, MOPS, KH2PO4, and KCl. http://dx.doi.org/10.1016/j.abb.2015.05.009 0003-9861/Ó 2015 Published by Elsevier Inc.

Alzheimer disease [4,5], multiple sclerosis [6], also cancer [7] and aging [8]. CypD is involved in the mechanism of permeability transition and in promotion of mitochondrial damage from Ca2+ overload [2,3,9–11]. Although, the physiological role of the PTP is well described, its structural constituents and the mechanistic aspects of its functioning remain elusive. Convincing evidence has been accumulated on bovine heart mitochondria which shows that the PTP is identical with dimers of F0/F1-ATPase [12–14] and that CypD is closely associated with the extrinsic part of the lateral stalk of the ATP synthase [15] being the binding site for the CypD inhibitor cyclosporin A (CsA) [16–19]. Furthermore, CsA interaction with CypD is phosphate mediated [20]. However, the exclusiveness of the binding mechanism remains unconfirmed. Very recently, involvement of phospholipase A2c as a pore component was suggested [21]. Some properties of PTP are tissue-specific [22–24]. The majority of CypD studies were performed on heart and liver mitochondria requiring distinct experimental conditions. Yet brain mitochondria possess a metabolic profile of their own. Particularly, they are more resistant to permeability transition than those of skeletal muscle [25,26] or liver [27,28]. This resistance correlates with a low

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expression of mitochondrial CypD in the brain as compared to skeletal muscle and liver [29]. This matches lower sensitivity to membrane depolarization and lower free radical generation in CypD deficient brain mitochondria as compared to the wild type mitochondria [30]. Consequently, CypD deficiency improves mitochondrial and synaptic function and learning/memory in mice [5]. It is intriguing to assume that CypD ablation is related to a reduced rate of neuronal apoptosis due to mitochondrial inability to activate PTP [2]. Therefore, CypD deficiency could render mitochondria more resistant against Ca2+ stress. However, CypD deficiency does not completely prevent mitochondria from permeability transition (PT) and from CsA inhibition [31]. Single-channel experiments on mouse liver mitochondria showed that lack of CypD simply shifts the affinity to CsA to higher concentrations [32]. There are a number of data indicating reciprocal control between PTP and respiratory complexes. They are controversial, possibly because of tissue-specific features of mitochondria. For example, in muscle cells CsA decreases ROS production after ischemia–reperfusion injury [33] and decreases complexes I and II-related respiration [34]. Long-term in vivo treatment of rats with CsA causes activation of state 3 respiration in liver mitochondria [35]. Furthermore, CsA causes both inhibition and uncoupling of respiration in kidney mitochondria [36]. In addition, CsA stimulates oxidative stress in renal tubular cells by decrease of antioxidant enzyme NAD(P)H and attenuation of the mitochondria membrane potential (DW) [37]. Bernardi’s group demonstrated that electron transport through complex I induces permeability transition during Ca2+ sequestration [26]. The same group showed that the modulation of CypD expression is the way cells regulate PT in response to inhibition of the complex I [38]. Rodriguez et al. found that long term exposure to CsA causes alterations in complex I activity of liver mitochondria [39]. Obviously the inhibitory effect of CsA on mitochondrial respiration is not well characterized and may be dependent on the tissue characteristics under investigation. Thus, tissue specific effects have to be taken in account when interpreting the experimental results. CypD can interact with both PTP and respiratory system and this interaction is tissue specific, too. In this respect brain mitochondria are not well characterized. In this work we aimed to study the involvement of CypD in regulation of energy metabolism of brain mitochondria under physiological conditions and under Ca2+ stress as well as in the presence of CypD inhibitor CsA. Experimental procedures Animals Experiments were performed on CypD knockout mice C57BL/6SV129 (Ppif / ) and their wild type (WT) analogues mice C57BL/6SV129 obtained from Howard Hughes Medical Institute, Children’s Hospital Medical Center, Cincinnati, OH, USA. CypD is encoded by the Ppif gene containing a mitochondrial targeting signal sequence that is cleaved after its translocation into the matrix, but the mature form contains a unique N terminus, which serves to identify CypD. The specifics of knock out Ppif were described previously [31]. Procedures for animal use were in accordance with the Animal Health and Care Committees of the Otto-von-Guericke University, Magdeburg, and of the State Sachsen-Anhalt, Germany. Isolation of mitochondria from mice brain Mixtures of non-synaptic and synaptic brain mitochondria from 12-week-old mice were isolated as described by Kudin et al. [40]. Immediately after decapitation, brains were rapidly removed and

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transferred into ice cold MMSE-A solution containing: 225 mM mannitol, 20 mM MOPS, 75 mM sucrose, 1 mM EGTA, 0.5 mM dithiothretol, pH 7.4. Further steps were performed at 4 °C. Tissue was minced and homogenized manually in ice cold MMSE-B solution, which is MMSE-A solution additionally containing 0.05% nagarse (1 g tissue/10 ml MMSE-B). A Dounce homogenizer was used applying four strokes with the pestle A (total clearance of 0.12 mm) and eight strokes with the pestle B (total clearance of 0.05 mm). Subsequently, 30 ml MMSE-A was added on top and the homogenate was centrifuged at 2,000g for 4 min at 4 °C. The supernatant was passed through cheesecloth and centrifuged at 12,000g for 10 min. For permeabilization of synaptosomal membrane and release the mitochondria the resulting pellet was resuspended in 10 ml MMSE-C (MMSE-A, additionally containing 0.02% digitonin) in a small glass homogenizer, and homogenized manually with 8–10 strokes to obtain a homogeneous suspension. This suspension was centrifuged at 12,000g for 10 min at 4 °C. The resulting pellet was resuspended in MMSE-D (225 mM mannitol, 20 mM MOPS, 75 mM sucrose, 0.1 mM EGTA, pH 7.4). Mitochondrial protein content was determined using the bicinchoninic acid assay [41]. Bovine serum albumin was used as a standard. Oxygen consumption Mitochondrial respiration was measured on high resolution OROBOROS oxygraph with a Clark-type oxygen electrode at 30 °C [42]. The oxygen concentration in an air-saturated medium was about 200 nmol O2/ml at 95 kPa. Weight-specific oxygen consumption was calculated from the time derivative of the oxygen concentration (DATGRAPH Analysis software, OROBOROS). Respiration of mitochondria (0.1 mg protein per ml) was measured in MMMPK medium containing 5 mM MgCl2, 120 mM mannitol, 20 mM MOPS, 5 mM KH2PO4, 60 mM KCl, pH 7.4, using a multiple substrates-inhibitor protocol as described previously [25,43], including 10 mM glutamate, 2 mM malate, 1.5 lM rotenone, 10 mM succinate, and 5 lM CAT. Mitochondrial membrane potential Alterations in energization of a mixture of synaptic and non-synaptic brain mitochondria (BM) were monitored fluorimetrically by recording the release of the membrane potential-sensitive probe safranine (2 lM), which is accumulated as a permeant cation in polarized BM. Fluorescence intensity was recorded at 495 nm excitation and 586 nm emission wavelengths using a Cary-Eclipse fluorescence spectrophotometer (Varian, Darmstadt, Germany) as described previously [44]. Cuvettes were stirred and thermostated at 30 °C. Membrane potential was measured in MMMPK buffer described above with 10 mM glutamate and 2 mM malate (glut/mal) as substrates. BM were added at a concentration of 0.1 mg protein/ml. Experiments were terminated by uncoupling of mitochondria with 1 lM FCCP. Ca2+ accumulation Extramitochondrial free Ca2+ was monitored with a Carry Eclipse fluorescence spectrophotometer (Varian, Darmstadt, Germany) using 0.5 lM Calcium Green-5 N at 506/532 nm excitation/emission wavelengths, respectively, in a stirred and thermostated (30 °C) cuvette as described previously [25]. The Ca2+ retention capacity (CRC) of mitochondria was measured by adding 10 lM Ca2+ aliquots in MMPK buffer containing 120 mM mannitol, 20 mM MOPS, 5 mM KH2PO4, 60 mM KCl, 10 lM ADP and 10 mM glutamate and 2 mM malate as substrates, pH 7.4. The mitochondria immediately started to accumulate Ca2+ causing a decrease

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of the fluorescence signal. The initial slope of this signal is proportional to the initial Ca2+ accumulation rate at the beginning of Ca2+ uptake [25]. When the fluorescence reached a stationary value, Ca2+ accumulation was assumed to be completed and the next Ca2+ addition was initiated. The 10 lM Ca2+ aliquots were added until the fluorescence started to increase, which was taken as an indication that Ca2+ was able to leave the mitochondria through the opened PTP. From these data the amount of Ca2+ which can be maximally accumulated until the PTP is completely opened, i.e. Ca2+ threshold ([Ca2+]thr), was calculated by equation [25]: [Ca2+]thr = [Ca2+]t  Fn, where [Ca2+]t is a total amount of the consumed Ca2+, which induces PT, multiplied by a sum of pulse-specific factors Fn. Fn accounts for the mitochondrial Ca2+ uptake during each Ca2+ pulse before PT occurs and equals the D Calcium Green-5 N fluorescence decrease during the nth Ca2+ uptake divided by the D Calcium Green-5 N fluorescence increase induced by the nth Ca2+ addition. The calcium accumulation indices were derived from the fluorescence spikes of the first Ca2+ pulse [25]: Ca2+ accumulation indices = rate of mitochondrial Ca2+ accumulation (tana of the slope of Calcium Green-5 N fluorescence decrease due to mitochondrial Ca2+ uptake)  pulse-specific completeness factor. Statistical analysis All results are presented as mean ± SD. Statistical analyses were performed using SigmaStat Software (SigmaPlot 11) for unpaired 2-tailed t test (for 2 groups). Results A functional network of mitochondria balances the processes of permeability transition and oxidative phosphorylation (OXPHOS). CypD, one of the components and regulatory proteins of PTP, is the mitochondrial target of CsA. To investigate the role of CypD in the complex interaction between the PT and OXPHOS systems we examined the functional properties of mitochondria isolated from whole brains of CypD KO versus WT mice under normal and Ca2+ stress condition with and without CsA. OXPHOS in brain mitochondria of CypD knockout mice To evaluate the functional activity of intact mitochondria isolated from the whole brain of wild type (BMWT) and CypD knockout mice (BMKO), the organelles were incubated in MMMPK medium with the complex I feeding substrates glutamate/malate. After the addition of low amounts of ADP (100 lM) OXPHOS was transiently activated. Under these conditions, the resting state of BMWT respiration (state 4glut/mal) was reached quickly due to the rapid phosphorylation of low amounts of ADP to ATP. Subsequently, application of 2 mM ADP led to a high rate of sustained respiration (state 3glut/mal). Addition of 1.5 lM rotenone caused a complete inhibition of complex I state 3glut/mal. The following application of succinate promoted complex II respiration (state 3suc). Finally, atractyloside (5 lM) was added to inhibit ADP entry through the adenine nucleotide exchanger enabling evaluation of the complex II state 4suc/atr. Under these experimental conditions, the rate of BMWT state 3glut/mal was similar to the rate of state 3suc, while the state 4glut/mal was lower than the state 4suc/atr (Fig. 1). The original respirograms could be found in Supplementary material. Thus, the corresponding respiratory control indices were proven to be different (RCIglut/mal = 3.08 ± 0.31; RCIsuc = 2.07 ± 0.07) (Table 1). Under the Ca2+ stress (in the presence of 20 lM Ca2+) the state 3glut/mal respiration of BMWT decreased by 53% as compared to the

control condition (Fig. 1). In contrast, the state 3suc decreased but shown to be statistically not significant. The state 3glut/mal/state 3suc ratio is an important indicator of the complex I-related metabolic impairments. The usefulness of this ratio to evaluate the complex I-mediated catabolism defects was described previously in [45]. This ratio was significantly lowered by Ca2+ addition (0.53 ± 0.02) as compared to the control value (0.82 ± 0.06) (Table 1). The complex I defects were also reflected by a 44% decrease of RCIglut/mal in the presence of 20 lM Ca2+, whereas the RCIsuc value was reduced tendentially only (Table 1). These respiratory data indicate impairment of BMWT complex I-dependent OXPHOS caused by Ca2+ overload. In case of CypD knockout mice mitochondria (BMKO), the state 3glut/mal respiration, RCIglut/mal and the ratio of state 3glut/mal/state 3suc were higher by 32%, 40%, and 40% respectively, than the corresponding values of BMWT (Fig. 1, Table 1). All together these parameters indicate that the level of the complex I-dependent OXPHOS of BMKO is remarkably higher than that of BMWT. However, the state 3suc of BMKO was very similar to that of BMWT (Fig. 1) demonstrating that the increased complex I-dependent OXPHOS is a result of specific changes, probably caused by ablation of CypD [13,14]. The Ca2+ stress decreased the BMKO state 3glut/mal respiration by 27% (Fig. 1). However, this effect was modest in comparison to the power by which Ca2+ decreased respiration of BMWT (by 53%). Comparison of BMKO respirogram in the presence of 20 lM Ca2+ (Supplementary material) with BMWT respirogram without Ca2+, as well as the corresponding mean values of the states 3glut/mal (Fig. 1) and RCIglut/mal (Table 1) revealed no significant difference between BMWT and BMKO indicating that even under Ca2+ stress mutant mitochondria have normal functional parameters similar to those of BMWT at physiological Ca2+ concentrations. The state 3suc respiration of BMKO in the presence of 20 lM Ca2+ remained unchanged similar to what was observed with BMWT. The increase of Ca2+ concentration up to 40 lM further reduced the rates of BMWT state 3glut/mal respiration by 70% with respect to the effect produced by 20 lM Ca2+. In contrast, the state 3glut/mal respiration of BMKO was not further decreased by the second addition of Ca2+. Finally, in the presence of a large concentration of Ca2+ (100 lM) the state 3glut/mal respiration was very low in both normal and mutant types of mitochondria indicating that the loss of CypD does not provide a complete protection of mitochondria from Ca2+ overload. Ablation of CypD rather shifts the dose-dependent response of BMKO to higher Ca2+ concentrations (data not shown). When 20 lM Ca2+ was applied in the presence of 1 lM CsA, the complex I-dependent respiration was nearly doubled compared to the incubation without CsA (Fig. 1). Also the RCI was only 13% less than the control but this value was decreased by 44% without CsA (Table 1). For comparison, we measured the effects of CsA alone on the functions of BMWT and BMKO. In BMWT under control conditions we observed a small insignificant increase of state 3glut/mal by 10% and nearly unchanged ratios of respiratory rates (Fig. 1, Table 1). In contrast to BMWT, pre-incubation of BMKO with 1 lM CsA decreased the state 3glut/mal respiration by 26% (Fig. 1) and RCI glut/mal by 30% (Table 1). The complex II-dependent respiration did not reveal any significant changes in the presence of CsA, neither in BMWT nor in BMKO. Another characteristic feature observed in BMKO in contrast to BMWT was that CsA did not change the effects of 20 lM Ca2+ on complex I-dependent OXPHOS. After application of 20 lM Ca2+ the RCIglut/mal value was 3.24 ± 0.33 versus 2.91 ± 0.36 in the presence of both Ca2+ and 1 lM CsA. As shown in Fig. 1 the rates of complex I and complex II-dependent respiration of BMKO, measured in the presence of 1 lM CsA, 20 lM Ca2+ or both together, did not differ from each other. Under all conditions applied, the

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Fig. 1. Respirometric properties of complex I- and complex II-dependent oxygen consumption of brain mitochondria from WT and CypD KO mice. Isolated BM (0.1 mg protein/ml) were investigated in MMMPK medium with 10 mM glutamate and 2 mM malate as substrates. To evaluate oxidative activity, the mitochondria were gradually challenged with 100 lM ADP to obtain complex I state 4, with 2 mM ADP to obtain complex I state 3, than with 10 mM succinate in the presence of 1.5 lM rotenone to obtain complex II state 3, and after addition of 5 lM atractyloside to obtain complex II state 4 (see Supplementary material). The quantitative data are presented for complex I state 3glut/mal, state 4glut/mal and complex II state 3rot/suc respirations under the control condition and in the presence of 1 lM CsA, 20 lM Ca2+ and both CsA and Ca2+ added 2 min before starting the recording. Data presented as mean ± SD (n = 4–6), #,§,ß,*p < 0.003, &,+p < 0.04.

Table 1 Control ratios of different respiration rates of brain mitochondria obtained in CypD knock-out (Ppif / ) versus wild type mice. Experimental parameters

WT

CypD KO

RCIglut/mal Control RCIglut/mal CsA RCIglut/mal Ca2+ RCIglut/mal CsA, Ca2+ RCIsuc Control RCIsuc CsA RCIsuc Ca2+ RCIsuc CsA, Ca2+ State 3glut/mal/State 3rot/suc State 3glut/mal/State 3rot/suc State 3glut/mal/State 3rot/suc State 3glut/mal/State 3rot/suc

3.08 ± 0.31#,& 3.20 ± 0.28 1.71 ± 0.11§,&,ß 2.67 ± 0.10ß 2.07 ± 0.07 1.67 ± 0.27 1.63 ± 0.18 1.88 ± 0.05 0.82 ± 0.06@,a 0.88 ± 0.08 0.53 ± 0.02⁄,a,u 0.80 ± 0.03u

5.03 ± 0.21#,+ 3.52 ± 0.37 3.24 ± 0.33§,+ 2.91 ± 0.36 2.40 ± 0.07 1.89 ± 0.06 2.09 ± 0.11 2.01 ± 0.24 1.36 ± 0.02@ 1.04 ± 0.10 1.07 ± 0.03⁄ 0.95 ± 0.08

Control CsA Ca2+ CsA, Ca2+

The experiments were performed under the conditions described in Fig. 1 in the presence and absence of CsA and Ca2+. Respiratory control indices (RCI) were calculated from the original respirograms (Supplementary material). RCIglut/mal is the state 3glut/mal/state 4glut/mal ratio measured after and before additions of 2 mM ADP, respectively. RCIsuc corresponds to the state 3suc/state 4atr recorded before and after the addition of 5 lM atractyloside (Atr) to succinate-respiring mitochondria. Results are shown as mean ± SD (n = 4–6), #,§,@,*, &,+,ß,,p < 0.003.

state 3glut/mal respiration of BMKO was by 20–28% lower than in control (no additions) while state 4glut/mal respiration remained unchanged. There were no changes observed in all succinate-dependent rates of respiration (Fig. 1). Membrane potential of brain mitochondria from CypD knockout mice The membrane potential (DW) of BM was tested fluorimetrically by recording the mitochondrial uptake and release of the membrane potential-sensitive probe safranine (2 lM). Mitochondria were incubated in MMMPK medium with glutamate/malate as substrates. Fig. 2 demonstrates representative original records of Ca2+-induced depolarization of both BMWT and BMKO and the same process in the presence of 1 lM CsA. In BMWT the addition of 10 lM Ca2+ immediately caused a remarkable release of safranine followed by reuptake and a new steady state with higher fluorescence than before the Ca2+ addition indicating a decrease of DW (Fig. 2A). After four Ca2+ additions nearly all safranine was released and, finally, the complete uncoupling

with FCCP resulted in further very small increase of fluorescence. The presence of 1 lM CsA (Fig. 2B and E) caused a significant reduction of the safranine fluorescence before challenging the BMWT with Ca2+, indicating that the DW values are clearly higher in mitochondria with CsA than in its absence (Fig. 2A, B and E). The mean values of safranine fluorescence of all measurements plotted on log scale against the applied Ca2+ concentrations are presented in Fig. 2E. In BMKO the initial safranine fluorescence values were much lower in comparison to those of BMWT (Fig. 2A, C and E). The first dose of Ca2+ induced a smaller release of safranine from BMKO than from BMWT. A total of 80 lM Ca2+ was required to induce nearly complete depolarization of BMKO, while in BMWT only 50 lM Ca2+ resulted in a collapse of DW. In contrast to BMWT, the decrease of DW in BMKO as a result of the exposure to Ca2+ was completely insensitive to 1 lM CsA (Fig. 2C–E). Ca2+ retention capacity and Ca2+ accumulation rates Next, we fluorimetrically analysed the effects of CypD ablation on the kinetic properties of mitochondrial Ca2+ accumulation using 0.5 lM Ca-Green-5N as an extramitochondrial Ca2+ sensor. Mitochondria were incubated in MMPK medium with glutamate/malate as substrates. As shown in Fig. 3A–D, addition of Ca2+ increased the fluorescence intensity of Ca-Green-5N. The BM immediately started to accumulate Ca2+ resulting in a decline of the fluorescence signal. The slope of the drop of the fluorescence following the Ca2+ additions is proportional to the rate of Ca2+ uptake by the mitochondria and can be quantified by means of the Ca2+ accumulation indices [24,25]. The addition of Ca2+ aliquot results in a maximal signal of Ca-Green-5N indicating the extramitochondrial concentration of Ca2+, which will then decline upon mitochondrial uptake of the added Ca2+ dose. The overload of mitochondria with Ca2+ that finally caused a complete PT was tracked by the onset of Ca2+ release from the mitochondria to the medium resulting in elevation of the fluorescence signal. Control BMWT were loaded with a total amount of 190 ± 5 lM Ca2+ by gradual addition of 10 lM Ca2+ aliquots per 0.1 mg mitochondrial protein/ml medium (Fig. 3A and E). Fig. 3B demonstrates the protective effect of 1 lM CsA on BMWT. The presence of CsA largely increased the rate of mitochondrial Ca2+ uptake by as much as 80% (Fig. 3A, B and F) and also increased the

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Fig. 2. Ca2+ modulation of the membrane potential in normal and mutant brain mitochondria. Brain mitochondria (0.1 mg/ml) of WT (A and B) and KO (C and D) mice were loaded with 2 lM safranine in MMPK buffer containing 10 mM glutamate and 2 mM malate, and measured with (B and D) and without (A and C) 1 lM CsA. Increased fluorescence emission values of safranine indicate membrane depolarization. Aliquots of 10 lM Ca2+ were added until no transient depolarization was further detectable. Finally, FCCP (1 lM) was added to induce complete depolarization of the mitochondria. (E) Dose–response curves for the Ca2+ additions were shifted to the higher concentrations in KO as compared to WT. In WT mitochondria CsA showed the protective effect against the pore opening since higher concentrations of Ca2+ were needed to provoke permeability transition. However, there was only a small difference in the amount of Ca2+ required for full depolarization of BMKO both with and without CsA. Data presented as mean ± SD (n = 4), *p < 0.04.

Fig. 3. Calcium retention capacity of brain mitochondria isolated from WT and KO mice. Fluorescence changes of extracellularly added 0.5 lM Calcium Green-5N were monitored in MMPK buffer containing 1 mg/ml mitochondria, 10 mM glutamate, 2 mM malate, and 10 lM ADP, either with (B and D) or without (A and C) 1 lM CsA. Aliquots of 10 lM Ca2+ were added as indicated. (E) Ca2+ accumulation threshold is linked to PT and thus to the beginning of mitochondrial Ca2+ release. (F) Calcium accumulation indices were obtained with and without CsA (see Experimental Procedures). Data presented as means ± SD from 4 independent experiments, #,§p < 0.002.

amount of Ca2+ that could be taken up by BMWT by 35% as compared with the control (Fig. 3A, B and E). Moreover, in the presence of CsA the Ca2+ uptake started and ended at much lower extramitochondrial Ca2+ as compared to the control conditions. As shown in Fig. 3A, C and F, the rate of Ca2+ uptake by BMKO was more than 85% higher than that by BMWT and the Ca2+ retention capacity of BMKO was significantly higher, too, than that of BMWT (by 30–40%). However, 1 lM CsA did neither increase the Ca2+ accumulation indices nor the Ca2+ threshold of BMKO (Fig. 3C, D and E).

Discussion The aim of this study was to explore the role of CypD in PTP operation and in oxidative phosphorylation processes of brain mitochondria. Impact of the lack of cyclophilin D on respiratory functions We comparatively studied the brain mitochondria isolated from WT and CypD KO (Ppif / ) animals. By three different approaches

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we demonstrated that under physiological Ca2+ concentrations CypD ablation results in improved functional properties of brain mitochondria. It was observed that the rates of glutamate/ malate-dependent OXPHOS were much higher in KO than in WT mitochondria. Elevated rates of the state 3glut/mal and unchanged state 4glut/mal respiration causing increased RCI values of BMKO were accompanied by increased values of membrane potential, rates of initial Ca2+ accumulation, and Ca2+ thresholds. These findings support the hypothesis that CypD regulates the PTP conformation by isomerization of pore proteins keeping the PTP always open to some extent [2,31,46]. The isolated normal mitochondria probably retain residual activity of CypD although in a down regulated state. In contrast, in BMKO CypD deficiency results in the lowest possible membrane conductivity with a higher DW leading to larger rates of state 3glut/mal respiration, as well as larger rates and amounts of Ca2+ uptake. Alternatively, the complex I metabolic constituents could also be directly influenced by CypD, having a higher activity if CypD is missing but being partially inhibited in its presence. The succinate-dependent respiration remained unaltered regardless of CypD presence pointing to a substrate specificity of CypD effects on respiration. The elevated membrane potential of BMKO cannot interfere with the state 3suc because mitochondrial succinate accumulation is an electroneutral process. In contrast DW can rather drive the glutamate/aspartate carrier which is an electrogenic process by nature [47] allowing an elevated state 3glut/mal in CypD deficient mitochondria. This effect is similar to acceleration of the initial Ca2+ accumulation in mutant brain mitochondria by higher DW (Fig. 2). Since CsA had generally no effect on succinate-dependent respiration, our results obtained on mitochondria missing CypD support the assumption that CsA could interact not only with its specific target CypD, but also with other constituents of the complex I related system. The important regulatory role of CypD was better uncovered by mitochondria exposure to Ca2+ stress. The addition of 20 lM Ca2+ to BMWT largely diminished the complex I but not complex II respiration (Fig. 1, Table 1). This impairment of the glutamate/malate-dependent metabolism worsened at further elevated Ca2+ concentrations. The effects of the Ca2+ stress on the state 3glut/mal of BMKO respiration were much less pronounced. However, at 100 lM Ca2+ BMKO also become completely impaired, indicating that very high Ca2+ amounts are capable of opening the PTP without CypD, too. Our results demonstrate that the mitochondria missing CypD acquire higher DW than WT mitochondria. Even after the addition of the first calcium dose the level of DW remains higher in BMKO than in BMWT (Fig. 2E). This clear difference shows that the Ca2+ induced decrease of DW is caused not only by an increase of DpH as a chemiosmotic consequence of Ca2+ uptake [48] but also by opening of PTP due to increased intramitochondrial Ca2+ content. Due to missing of CypD, which is a Ca2+ sensitive protein, the DW of BMKO is less responsive to Ca2+ stress. On the other hand, high Ca2+ concentration can cause a complete loss of membrane potential of BMKO, too (Fig. 2). The direct inhibition of complex I by Ca2+ is not excluded, however earlier we demonstrated that the enzymatic activity of complex I is not influenced by increasing doses of Ca2+ in accord with the recent data of Pandya [49] (data not shown). Differential CsA effects on BMWT and BMKO CsA is known to inhibit the pore opening effect of CypD, although the exact mechanism of CsA and CypD interaction is still under debate [2,12,13,38,50]. In this work we showed that 1 lM CsA moderately (statistically non-significant) increased the rate of complex I state 3 respiration and RCIglut/mal of BMWT (Fig. 1).

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This tendency to magnify mitochondria respiratory activity is in line with our previous findings showing that CsA (2 lM) elevates the state 3 respiration of BM isolated from 6 different brain regions [25]. In agreement with these recent results, CsA also increased mitochondrial membrane potential, the rates of initial Ca2+ accumulation, as well as Ca2+ thresholds in BMWT (Figs. 2 and 3). These effects can be explained by closing of PTP due to CsA binding with CypD. In full agreement with this theory, CsA did not affect the membrane potential, initial Ca2+ accumulation rates and Ca2+ thresholds in BMKO but inhibited (although non-significantly) the state 3glut/mal of BMKO (Figs. 2 and 3). Therefore, in BMKO missing the CsA target CypD the preferable conformation of PTP is that favoring its closed state. However, how to explain inhibition of the state 3glut/mal but not of the state 3suc of BMKO? It cannot be explained by CsA inhibition of mitochondrial glutamate catabolism proteins, such as glutamate/aspartate carrier or complex I, because in this case a decreased flux through this metabolic port, which builds up the membrane potential, should be an essential consequence. Possibly, CsA are capable of interacting with other mitochondrial targets altering the OXPHOS by inhibition of both DW-generating and DW-utilizing reactions. Conclusions The data obtained in this study using a genetically ablated Ppif / mouse model demonstrated the critical role of CypD in control of the oxidative phosphorylation processes along with the control of PTP in brain mitochondria. The results provide evidence for the direct involvement of respiratory system in mechanisms of permeability transition in brain mitochondria. Since the knockout of CypD results in improved functional properties of brain mitochondria, than this should be an additional reason to regard CsA or its non-immunosuppressive derivative Cs9 [25,51] as potential pharmaceutical drugs to protect mitochondrial function from Ca2+ stress. Acknowledgments The support of Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) and Drexel University Professional Enrichment and Growth Grant (to Z.O.) are appreciated. We thank Richard Sensenig for help with manuscript preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2015.05.009. References [1] G. Fischer, B. Wittmann-Liebold, K. Lang, T. Kiefhaber, F.X. Schmid, Nature 337 (1989) 476–478. [2] C.P. Baines, R.A. Kaiser, N.H. Purcell, N.S. Blair, H. Osinska, M.A. Hambleton, E.W. Brunskill, M.R. Sayen, R.A. Gottlieb, G.W. Dorn, J. Robbins, J.D. Molkentin, Nature 434 (2005) 658–662. [3] A.C. Schinzel, O. Takeuchi, Z. Huang, J.K. Fisher, Z. Zhou, J. Rubens, C. Hetz, N.N. Danial, M.A. Moskowitz, S.J. Korsmeyer, PNAS 102 (2005) (2010) 12005– 12010. [4] L. Guo, H. Du, S. Yan, X. Wu, G.M. McKhann, J.X. Chen, S.S. Yan, PloS One 8 (2013) e54914. [5] H. Du, L. Guo, W. Zhang, M. Rydzewska, S. Yan, Neurobiol. Aging 32 (2011) 398–406. [6] M. Forte, B.G. Gold, G. Marracci, P. Chaudhary, E. Basso, D. Johnsen, X. Yu, J. Fowlkes, M. Rahder, K. Stem, P. Bernardi, D. Bourdette, PNAS 104 (2007) 7558– 7563. [7] K. Machida, Y. Ohta, H. Osada, J. Biol. Chem. 281 (2006) 14314–14320. [8] D. Brust, B. Daum, C. Breunig, A. Hamann, W. Kuhlbrandt, H.D. Osiewacz, Aging cell 9 (2010) 761–775.

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