Cyclophilin D in Mitochondrial Pathophysiology

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Biochimica et Biophysica Acta 1797 (2010) 1113–1118

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbabio

Review Cyclophilin D in mitochondrial pathophysiology

Valentina Giorgio a, Maria Eugenia Soriano b, Emy Basso a, Elena Bisetto c, Giovanna Lippe d, Michael A. Forte e, Paolo Bernardi a,b,⁎

a Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Italy b Venetian Institute of Molecular Medicine, Padova, Italy c Department of Biomedical Sciences and Technologies, University of Udine, Italy d Department of Food Science, University of Udine, Italy e Vollum Institute, Oregon Health and Sciences University, Portland, Oregon, USA

article info abstract

Article history: Cyclophilins are a family of peptidyl-prolyl cis–trans isomerases whose enzymatic activity can be inhibited Received 6 November 2009 by cyclosporin A. Sixteen cyclophilins have been identiﬁed in humans, and cyclophilin D is a unique isoform Received in revised form 27 November 2009 that is imported into the mitochondrial matrix. Here we shall (i) review the best characterized functions of Accepted 4 December 2009 cyclophilin D in mitochondria, i.e. regulation of the permeability transition pore, an inner membrane channel Available online 21 December 2009 that plays an important role in the execution of cell death; (ii) highlight new regulatory interactions that are emerging in the literature, including the modulation of the mitochondrial F1FO ATP synthase through an Keywords: Cyclophilin interaction with the lateral stalk of the enzyme complex; and (iii) discuss diseases where cyclophilin D plays Mitochondria a pathogenetic role that makes it a suitable target for pharmacologic intervention. ATP synthase © 2009 Elsevier B.V. All rights reserved. Permeability transition Cyclosporin A

1. Introduction from CsA binding and calcineurin inhibition [9], suggesting that CyPs have specific cellular functions that may be of importance for a variety Cyclophilins (CyPs) are ubiquitous proteins endowed with of processes relevant to human disease [3]. peptidyl-prolyl cis–trans isomerase (PPIase) activity [1,2] that have The evolutionary conservation of the PPIase activity among species been highly conserved during evolution. CyPs can be identified in the suggests that this can be an important function of the CyPs and FKBPs genomes of mammals, plants, insects, fungi and bacteria; they all [10], as shown by the Drosophila NinaA PPIase, which serves as a share a common domain of approximately 109 amino acids, the CyP- chaperone for specific rhodopsin isoforms [11]. Yet, and somewhat like domain [3]. In humans 16 unique CyPs have been found [3], with surprisingly, Saccharomyces cerevisiae mutants lacking all 12 yeast CyPA representing the prototype of the family [1,2]. After binding to immunophilins were viable, and the phenotype of the dodecuplet the CyP ligand cyclosporin (Cs) A, the PPIase activity is inhibited mutant resulted from simple addition of the subtle phenotypes of [4], and the CsA/CyPA complex binds to and inhibits the cytosolic each individual mutation [12]. This striking finding led these authors phosphatase calcineurin [5] resulting in immunosuppression [6,7]. to conclude that CyPs and FKBPs do not play an essential general role Together with the FK506-binding proteins (FKBP, structurally in protein folding, and to propose that each CyP and FKBP instead may unrelated PPIases that share with CyPs the ability to inhibit regulate a restricted number of unique partner proteins [12].In calcineurin after binding their cognate inhibitory ligand FK506), keeping with this prediction, CyPs have been shown in recent years to they constitute the family of immunophilins [8]. Work with mutants be involved in a variety of pathophysiological processes including of human CyPA has clearly separated the PPIase activity of the protein inflammation and vascular dysfunction [13–17], wound healing [18], innate immunity to HIV [19], hepatitis C infection [20], host–parasite interactions [21], tumor biology [22] and regulation of the mitochondrial permeability transition pore (PTP) which is mediated by the Abbreviations: ANT, adenine nucleotide translocator; Cs, cyclosporin; CyP, cyclophilin; mitochondrial isoform of the enzyme, CyPD [23–26]. The existence of FKBP, FK506-binding proteins; PPIase, peptidyl-prolyl cis–trans isomerase; PTP, specific functions is also suggested by the presence of tissue- and permeability transition pore organelle-specific isoforms characterized by the combination of the ⁎ Corresponding author. Department of Biomedical Sciences, University of Padova, Viale Giuseppe Colombo 3, I-35121 Padova, Italy. Fax: +39 049 827 6361. signature CyP domain with the proper targeting and/or retention E-mail address: [email protected] (P. Bernardi). sequence(s) [3].

2. Cyclosporin A, cyclophilins and calcineurin of PTP inhibition by CsA and CyPD ablation may be just one example of the factors that control the PTP sensitivity to inhibitors in situ. Thus, Cs are cyclic undecapeptides produced by several fungal taxa extreme caution should be exerted when results from in vitro studies including the common strain Tolypocladium inflatum. CsA is the most are extrapolated to the status of the PTP in vivo. Fig. 1 presents a studied [27]; its ability to prevent the immune response has scheme where the effects of CyPD and Pi on the PTP are summarized. revolutionized medicine, allowing organ transplantion to become a In the CyPD-bound state the Pi site is not available, and the PTP favors standard therapeutic practice. Interaction of CsA with cytosolic CyPA the open conformation (right). Addition of CsA (or CyPD ablation) generates a complex that acquires the ability to bind to, and inhibit, unmasks the Pi binding site by removing CyPD (middle); if the free the cytosolic, Ca2+-activated phosphatase calcineurin [5];asa [Pi] concentration is high enough [50], Pi binding to the PTP lowers consequence of calcineurin inhibition, its substrate phospho-NFAT is the open probability (left) in a reaction that is readily reversible if the no longer dephosphorylated and therefore unable to translocate to [Pi] concentration decreases. Although no endogenous ligands of the nucleus and trigger the IL-2-dependent activation of the immune CyPD mimicking the effects of CsA are currently known, other PTP response against the transplant [6,7]. Recently, an important (de)sensitizers might act by favoring CyPD association or dissociation. discovery has been made showing that translocation of the cytosolic Indeed, the existence of additional effector sites on the PTP is strongly pro-fission protein Drp1 to mitochondria requires its calcineurin- suggested by the large variety of PTP inducers and inhibitors [51]. dependent dephosphorylation at Ser637 that is inhibited by CsA [28]. Thus, CsA also affects mitochondrial shape and function via calci- 4. Cyclophilin D interactions with other proteins neurin inhibition independent of its interactions with matrix CyPD (see below), a concept that should be taken into account when An interaction of CyPD with the adenine nucleotide translocator interpreting the complex effects of CsA on mitochondrial function and (ANT) has been described both in detergent extracts of mitochondria cell survival [29]. Separation of the calcineurin-dependent and and after purification of the ANT [52]. These results have been -independent effects of Cs has been greatly helped by non- interpreted within the framework of the PTP being formed by the ANT immunosuppressive analogs of CsA (such as MeVal-4-Cs, NIM811 and regulated by CyPD binding [53], possibly with the contribution of and Debio 025) that maintain the ability to bind to, and inhibit CyPs VDAC as well [54]. As we have noted previously [55], in these but not calcineurin [24,30–32]. Like Cs these drugs likely inhibit all protocols several proteins other than the ANT bound to CyPD in a CsA- CyPs to varying degrees [3,26], a point that should be kept in mind inhibitable manner [52]. Notably, CyPD bound equally well to ANT when the mitochondrial effects of these molecules are evaluated. A purified from rat liver and yeast, yet the permeability transition is not recent study has identified a small-molecule inhibitor based on aryl 1- inhibited by CsA in yeast mitochondria [56]. Our concerns about the indanylketones that discriminates between CyPA and CyPB in vitro,a significance of the CyPD–ANT(-VDAC) interactions in PTP regulation proof of principle that holds great promise for the development of are reinforced by the demonstration that ANT-null mitochondria still isoform-specific CyP inhibitors [33]. The rapidly growing field of CyP display a CsA-sensitive permeability transition, although this requires biochemistry, pharmacology and pathophysiology is beyond the higher loads of Ca2+ and Pi [57], and that VDACs are dispensable for scope of this article, but the interested reader can find further PTP opening [58,59]. It should be noted that the identification of the information in recent reviews [3,34,35]. ANT as a CyPD-binding protein was obtained by immunological methods rather than by direct protein determination by sequencing 3. Cyclophilin D and the permeability transition or mass spectroscopy [52]. Recent work has shown that the polyclonal antibody used in these studies is not detecting ANT1 but rather the Pi CyPD is the unique mitochondrial isoform of CyP in mammals [23– carrier [60], which has now been incorporated in the model of the PTP 25]. Its identification was prompted by the demonstration that CsA proposed by the Halestrap laboratory [61], a working hypothesis that affects mitochondrial Ca2+ fluxes [36] through an effect that could be in our opinion needs to be validated by more stringent tests. traced to desensitization of the PTP [37–40], an inner membrane high- Further regulatory interactions of CyPD are emerging in the conductance channel whose molecular composition remains an literature. A recent study has demonstrated the existence of a unanswered question [41–45]. The PTP plays a key role in cell death complex mitochondrial chaperone network that involves Hsp90, its and has proved to be a viable target for therapeutic intervention in a related molecule TRAP-1, and CyPD [62]. CyPD bound to the complex variety of diseases, most notably ischemia–reperfusion injury of the would no longer be available for PTP opening. CyPD displacement heart and brain, muscular dystrophies, neurodegeneration and cancer [41–45]. Genetic ablation of the Ppif gene (which encodes for CyPD) in the mouse has demonstrated that CyPD is the mitochondrial receptor for CsA, and that it is responsible for modulation of the PTP but not a structural pore component [46–49]. As discussed more in detail elsewhere [42], the effect of CsA on the PTP is best described as “desensitization” in the sense that the PTP becomes more resistant to opening after the uptake of Ca2+ and Pi in standard in vitro assays in isolated mitochondria; yet pore opening readily takes place for Ca2+– Pi loads that are about twice those required in wild-type mitochondria. A major step forward in our mechanistic understanding of the role of CyPD in PTP modulation has been the discovery that CyPD ablation (or treatment with CsA) unmasks an inhibitory site for Pi, which is the actual PTP desensitizing agent [50]. Unless Pi is present, the sensitivity of the PTP to Ca2+ and to other agents of pathophysiological relevance is identical in naïve and CsA-treated wild type mitochondria, as well as in CyPD-null mitochondria. This finding has important implications for our understanding of PTP regulation. Fig. 1. Modulation of the permeability transion pore by CyPD and Pi. The CyPD-bound form of the PTP displays a higher probability of opening (right); addition of CsA Indeed, as also noted elsewhere [50] (i) it is fortunate that Pi was displaces CyPD and unmasks a cryptic site for Pi (center); if the Pi concentration is included in mitochondrial swelling assays of PTP in vitro, so that the sufficiently high, Pi binding decreases the probability of pore opening (left), in a desensitizing effect of CsA was not missed; and (ii) the Pi-dependence reaction that can be readily reversed if the Pi concentration decreases. V. Giorgio et al. / Biochimica et Biophysica Acta 1797 (2010) 1113–1118 1115 from this complex by selective Hsp90 antagonists like Shepherdin could disable this protective network and reactivate the PTP with the onset of mitochondrial depolarization, release of cytochrome c, and massive cell death. Since the Hsp90–TRAP-1–CyPD interactions are particularly prominent in tumor cells, this strategy has been suc- cessfully used for their selective killing [62]. In keeping with previous results on the role of GSK-3 in PTP regulation [63], we have recently found that a small fraction of ERK associates to mitochondria, where it can be activated to phosphor- ylate (hence inactivate) GSK-3 resulting in PTP desensitization. Of note, CyPD could be co-immunoprecipitated with ERK2 and GSK-3, and it was Ser/Thr phosphorylated. Phosphorylation correlated with CyPD binding to GSK-3 and with PTP opening; and both events were inhibited by the selective GSK-3 inhibitor indirubin-3′-oxime [64]. A recent study has demonstrated an interaction of CyPD with Bcl2 that could be displaced by CsA. In these protocols, addition of CsA increased tBid-dependent release of cytochrome c from mitochondria Fig. 2. Interactions of CyPD with the lateral stalk of the F1FO ATP synthase. The ATP under conditions that did not cause the opening of the PTP, suggesting synthase is depicted in the dimeric form, and the drawing highlights the possible site of a PTP-independent effect of CyPD (and CsA) that is relevant to interaction of CyPD with the lateral stalk, possibly at subunit b. For further explanation mitochondrial triggering of apoptosis [65]. Consistent with previous see text. results [66], CyPD overexpression made cells more resistant to apoptotic stimuli, a finding that is difficult to reconcile with a predominant effect of overexpression on the PTP [65,66]. stimulatory effect of CsA is lost in Ppif−/− mitochondria, indicating 5. Cyclophilin D interactions with the F1FO ATP synthase that it is mediated by CyPD, while assembly of the ATP synthase is unaffected by CyPD ablation [69]. The F1FO ATP synthase is a 600 kDa multisubunit complex located in the inner mitochondrial membrane whose catalytic part (F1) and lateral stalk protrude into the matrix compartment where CyPD is 6. Cyclophilin D in disease located, while the FO moiety and the remaining portion of the lateral stalk are embedded into the inner membrane [67,68]. We recently Ppif−/− mice do not display an overt disease phenotype [46–49].In documented that CyPD interacts with the ATP synthase of bovine a number of independent laboratories, CyPD-null mice were born at heart mitochondria in protocols based on both blue native electro- the expected Mendelian ratios [46–49] and were apparently identical phoresis and immunoprecipitation of complex V after mild detergent to wild-type, Ppif+/+ C57BL/6 animals, findings that indicate that extraction [69]. Cross-linking with the cleavable bifunctional reagent CyPD is dispensable for embryonic development and viability [47].It dimethyl 3,3-dithiobis-propionimidate, which reacts with the prima- is possible that lack of a phenotype at birth may be due to adaptive ry amines of two interacting proteins at an average distance of about responses whereby the decreased sensitivity of the PTP to Ca2+ is 8Å[70], demonstrated that CyPD forms a cross-linked complex with bypassed by compensatory mechanisms that are not detectable in OSCP, b, and d subunits in an apparent ratio of 1:1:1:1 [69], supporting isolated mitochondria. It should also be noted that the physiological the interaction of CyPD with the extrinsic part of the lateral stalk. The role of CyPD may change during development, as neonatal Ppif−/− cross-linker also forms smaller complexes (such as b–d and b–OSCP) mice are more sensitive to ischemia–reperfusion injury of the brain [71], and so far we have been unable to resolve the CyPD direct [75] rather than more resistant as are adult mice [49,75]. However, interactor(s) on the ATP synthase. Since only subunit b was detected Ppif−/− mice display increased anxiety, facilitation of avoidance amongst CyPD-binding proteins in affinity purification-based proto- behavior, and they develop adult-onset obesity independent of food cols (C. Baines, personal communication), we suspect that the direct and water intake [76] suggesting that they may develop specific interaction of CyPD may occur with the surface area of subunit b phenotypes with aging. It should also be recalled that CyPD exposed to the mitochondrial matrix [67]. It is important to stress that overexpression desensitized cells from apoptotic stimuli, indicating interaction of CyPD with the enzyme complex is favored by Pi and that CyPD may also play a role as a cell survival-signaling molecule competed by CsA. Importantly, CyPD binding has major functional acting on targets other than the PTP [65,66]. consequences, with a decrease of specific activity that can be reversed CsA and its non-immunosuppressive analogs MeVal-4-CsA, by CsA-dependent CyPD displacement [69]. Whether this striking NIM811 and Debio 025 have been used in disease paradigms where analogy with Pi-dependent PTP regulation by CyPD [50] indicates a the PTP has been causally implied such as ischemia–reperfusion injury link between the two processes remains a matter for future studies. of the heart and brain, TNF-α-dependent fulminant hepatitis, and Fig. 2 illustrates the putative site(s) of interaction of CyPD with the collagen VI myopathies (reviewed in [42,77]). As should be apparent lateral stalk of the F1FO ATP synthase, depicted here in the dimeric from the preceding paragraphs, the interpretation of these results is form, which is considered the “building block” of higher order, not straightforward because (i) all CyPs, not only CyPD, are likely oligomeric forms of the enzyme that optimize its catalytic perfor- inhibited to varying degrees by these drugs; (ii) the recent finding mance; oligomeric forms are considered to represent the physiolog- that Drp1 dephosphorylation depends on calcineurin [28] demands a ical state of F1FO ATP synthase in the inner membrane [72,73]. There reinterpretation of the protective effects of CsA in many animal is a wide variability in the angle formed by the monomers when they models of human disease since CsA's mitochondrial effects may in associate into dimers [74], and it is tempting to speculate that CyPD part depend on inhibition of Drp1-mediated fission; and (iii) the PTP displacement by CsA may activate the enzyme by favoring the is not directly affected by CsA but rather desensitized through the formation of oligomers characterized by a higher activity. Conversely, inhibitory effects of Pi, and the PTP is insensitive to CsA if the Pi CyPD binding could switch the enzyme to a different oligomeric form concentration is not high enough [50]. The only models that allow (i.e. a state with a different angle between monomers), with lower conclusions to be made on the role of CyPD in disease are those based catalytic activity [69]. Finally, it should be mentioned that the on the genetic ablation of CyPD (Ppif−/− mice), with the clear 1116 V. Giorgio et al. / Biochimica et Biophysica Acta 1797 (2010) 1113–1118

Table 1 inhibitors of increasing specificity in CyPD-dependent diseases. In −/− Role of CyPD in mouse disease models as assessed in Ppif animals. addition, the emerging complexity of the mitochondrial effects of Disease model Effect Reference(s) CyPD (hence of CsA) should induce some caution in interpreting the results obtained with CyP inhibitors and CyPD ablation as solely Heart ischemia–reperfusion Protection [46,48] Brain ischemia–reperfusion resulting from modulation of the PTP. Adult Protection [49,75] Neonatal Worsening [75] Acknowledgements Autoimmune encephalomyelitis Protection [86] Mutant amyloid precursor protein mice Protection [88] (Alzheimer's disease) This work was supported by Grants from the Fondazione Cariparo Superoxide dismutase 1 G93A mutant mice Protection [87] (Padova), the University of Padova Progetti di Eccellenza “Models of (amyotrophic lateral sclerosis) mitochondrial diseases” and Telethon - Italy (to PB), and by the NIH- −/− Scgd (muscular dystrophy linked to Protection [99] PHS (grant GM69883 to MAF and PB). sarcoglycan deficiency) Lama2−/− (muscular dystrophy linked to Protection [99] laminin deficiency) References Col6a1−/− (muscular dystrophy linked to Protection [96] collagen VI deficiency [1] G. Fischer, B. Wittmann-Liebold, K. Lang, T. Kiefhaber, F.X. Schmid, Cyclophilin and peptidyl-prolyl cis–trans isomerase are probably identical proteins, Nature 337 (1989) 476–478. [2] N. Takahashi, T. Hayano, M. Suzuki, Peptidyl-prolyl cis–trans isomerase is the understanding that this animal model explores the role of CyPD, not of cyclosporin A-binding protein cyclophilin, Nature 337 (1989) 473–475. [3] P. Wang, J. Heitman, The cyclophilins, Genome Biol. 6 (2005) 226.1–226.6. the PTP, in the considered disease paradigm. [4] J.F. Borel, C. Feurer, C. Magnee, H. Stahelin, Effects of the new anti-lymphocytic −/− Convincing evidence obtained in adult Ppif mice causally links peptide cyclosporin A in animals, Immunology 32 (1977) 1017–1025. CyPD to ischemia–reperfusion injury of the heart [46,48] and brain [5] J. Liu, J.D.J. Farmer, W.S. Lane, J. Friedman, I. Weissman, S.L. Schreiber, Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK506 complexes, Cell [49,75], as infarct size was reduced in CyPD-null adults compared to 66 (1991) 807–815. wild-type or littermate controls. These studies corroborate the [6] N.A. Clipstone, G.R. Crabtree, Identification of calcineurin as a key signalling conclusions of previous studies based on the use of CsA in ischemic enzyme in T-lymphocyte activation, Nature 357 (1992) 695–697. isolated hearts [78] and in infarcted patients [79], in brain damage by [7] C.T. Walsh, L.D. Zydowsky, F.D. McKeon, Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction, J. Biol. Chem. hypoglycemia [80], hyperglycemia [81], middle cerebral artery 267 (1992) 13115–13118. occlusion [82,83], and traumatic injury [84,85]. Ppif−/− mice [8] A. Galat, Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity— — – displayed a striking resistance to development of axonopathy in a targets functions, Curr. Top. Med. Chem. 3 (2003) 1315 1347. [9] L.D. Zydowsky, F.A. Etzkorn, H.Y. Chang, S.B. Ferguson, L.A. Stolz, S.I. Ho, C.T. Walsh, model of autoimmune encephalomyelitis [86],andtodisease Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase progression after crossing with superoxide dismutase 1 mutant activity from cyclosporin A binding and calcineurin inhibition, Protein Sci. 1 – mice [87], suggesting that CyPD-dependent mechanisms may be (1992) 1092 1099. [10] E.R. Schonbrunner, S. Mayer, M. Tropschug, G. Fischer, N. Takahashi, F.X. Schmid, critical in the neurodegenerative aspects of demyelinating and motor Catalysis of protein folding by cyclophilins from different species, J. Biol. Chem. neuron diseases. Ablation of CyPD has also been shown to substan- 266 (1991) 3630–3635. tially improve learning, memory and synaptic function in a mouse [11] M.A. Stamnes, B.H. Shieh, L. Chuman, G.L. Harris, C.S. Zuker, The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the model of Alzheimer's disease, where it also alleviated reduction of proper synthesis of a subset of Drosophila rhodopsins, Cell 65 (1991) 219–227. long-term potentiation mediated by the amyloid β peptide [88]. [12] K. Dolinski, S. Muir, M. Cardenas, J. Heitman, All cyclophilins and FK506 binding The best characterized case involving CyPD in pathology in vivo is proteins are, individually and collectively, dispensable for viability in Saccharo- myces cerevisiae, Proc. Natl Acad. Sci. USA 94 (1997) 13093–13098. represented by collagen VI diseases [89], a set of genetically [13] Z.G. Jin, A.O. Lungu, L. Xie, M. Wang, C. Wong, B.C. Berk, Cyclophilin A is a heterogenous conditions that cause Bethlem myopathy [90], Ullrich proinflammatory cytokine that activates endothelial cells, Arterioscler. Thromb. congenital muscular dystrophy [91,92] and myosclerosis [93] in Vasc. Biol. 24 (2004) 1186–1191. humans. Following the discovery that the mouse model of the disease [14] S.H. Kim, S.M. Lessner, Y. Sakurai, Z.S. Galis, Cyclophilin A as a novel biphasic mediator of endothelial activation and dysfunction, Am. J. Pathol. 164 (2004) −/− (Col6a1 lacking collagen VI [94]) has a striking mitochondrial 1567–1574. involvement in affected muscles that could be cured with CsA [95] and [15] H. Kim, W.J. Kim, S.T. Jeon, E.M. Koh, H.S. Cha, K.S. Ahn, W.H. Lee, Cyclophilin A fl Debio 025 [32], we have recently shown that Ppif−/− Col6a1−/− mice may contribute to the in ammatory processes in rheumatoid arthritis through induction of matrix degrading enzymes and inflammatory cytokines from lacking both CyPD and collagen VI are indistinguishable from syngenic macrophages, Clin. Immunol. 116 (2005) 217–224. wild type mice [96], providing a clear proof of principle for the causal [16] K. Arora, W.M. Gwinn, M.A. Bower, A. Watson, I. Okwumabua, H.R. MacDonald, M. involvement of CyPD in the development of the disease that matches I. Bukrinsky, S.L. Constant, Extracellular cyclophilins contribute to the regulation of inflammatory responses, J. Immunol. 175 (2005) 517–522. the promising results obtained with CsA in patient cultures [97] and in [17] J.M. Damsker, M.I. Bukrinsky, S.L. Constant, Preferential chemotaxis of activated a pilot trial [98], and with the non-immunosuppressive CyP inhibitor human CD4+ T cells by extracellular cyclophilin A, J. Leukoc. Biol. 82 (2007) Debio 025 in the collagen VI myopathic mouse [32]. Although the 613–618. [18] W. Kong, S. Li, M.T. Longaker, H.P. Lorenz, Cyclophilin C-associated protein is up- therapeutic outcome was less dramatic, these results are matched by regulated during wound healing, J. Cell Physiol. 210 (2007) 153–160. −/− −/− those obtained in Ppif Scgd mice, which showed markedly less [19] E. Sokolskaja, J. Luban, Cyclophilin, TRIM5, and innate immunity to HIV-1, Curr. dystrophic disease in both skeletal muscle and heart than Scgd−/− Opin. Microbiol. 9 (2006) 404–408. δ −/− −/− [20] R. Flisiak, J.M. Dumont, R. Crabbe, Cyclophilin inhibitors in hepatitis C viral mice lacking -sarcoglycan [99]. In addition, Ppif Lama2 mice infection, Expert. Opin. Investig. Drugs 16 (2007) 1345–1354. −/− exhibited a significantly extended survival compared to Lama2 [21] A. Bell, P. Monaghan, A.P. Page, Peptidyl-prolyl cis–trans isomerases (immuno- mice and treatment of the mdx mouse model of Duchenne muscular philins) and their roles in parasite biochemistry, host–parasite interaction and – dystrophy with Debio 025 resulted in markedly less dystrophic antiparasitic drug action, Int. J. Parasitol. 36 (2006) 261 276. [22] Q. Yao, M. Li, H. Yang, H. Chai, W. Fisher, C. Chen, Roles of cyclophilins in cancers disease [99]. The mouse disease models in which the effects of CyPD and other organ systems, World J. Surg. 29 (2005) 276–280. ablation have been investigated are summarized in Table 1. [23] C.P. Connern, A.P. Halestrap, Purification and N-terminal sequencing of peptidyl- prolyl cis–trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin, Biochem. J. 284 (1992) 381–385. [24] A. Nicolli, E. Basso, V. Petronilli, R.M. Wenger, P. Bernardi, Interactions of cy- 7. Conclusions clophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, a cyclosporin A-sensitive channel, J. Biol. Chem. 271 (1996) 2185–2192. We think that understanding the pathophysiology of CyPD will [25] K.Y. Woodfield, N.T. Price, A.P. Halestrap, cDNA cloning of rat mitochondrial provide novel perspectives to the development and use of CyP cyclophilin, Biochim. Biophys. Acta 1351 (1997) 27–30. V. Giorgio et al. / Biochimica et Biophysica Acta 1797 (2010) 1113–1118 1117

[26] P.C. Waldmeier, K. Zimmermann, T. Qian, M. Tintelnot-Blomley, J.J. Lemasters, [55] E. Fontaine, P. Bernardi, Progress on the mitochondrial permeability transition Cyclophilin D as a drug target, Curr. Med. Chem. 10 (2003) 1485–1506. pore. Regulation by Complex I and ubiquinone analogs, J. Bioenerg. Biomembr. 31 [27] J.F. Borel, C. Feurer, H.U. Gubler, H. Stahelin, Biological effects of cyclosporin A: a (1999) 335–345. new antilymphocytic agent, Agents Actions 6 (1976) 468–475. [56] D.W. Jung, P.C. Bradshaw, D.R. Pfeiffer, Properties of a cyclosporin-insensitive [28] G.M. Cereghetti, A. Stangherlin, O. Martins de Brito, C.R. Chang, C. Blackstone, P. permeability transition pore in yeast mitochondria, J. Biol. Chem. 272 (1997) Bernardi, L. Scorrano, Dephosphorylation by calcineurin regulates translocation of 21104–21112. Drp1 to mitochondria, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 15803–15808. [57] J.E. Kokoszka, K.G. Waymire, S.E. Levy, J.E. Sligh, J. Cai, D.P. Jones, G.R. MacGregor, [29] M. Wasilewski, L. Scorrano, The changing shape of mitochondrial apoptosis, D.C. Wallace, The ADP/ATP translocator is not essential for the mitochondrial Trends Endocrinol. Metab. 20 (2009) 287–294. permeability transition pore, Nature 427 (2004) 461–465. [30] G. Baumann, G. Zenke, R. Wenger, P. Hiestand, V. Quesniaux, E. Andersen, M.H. [58] A. Krauskopf, O. Eriksson, W.J. Craigen, M.A. Forte, P. Bernardi, Properties of the Schreier, Molecular mechanisms of immunosuppression, J. Autoimmun. 5 (Suppl permeability transition in VDAC1−/− mitochondria, Biochim. Biophys. Acta A) (1992) 67–72. Bioenergetics 1757 (2006) 590–595. [31] M.J. Hansson, G. Mattiasson, R. Mansson, J. Karlsson, M.F. Keep, P. Waldmeier, U.T. [59] C.P. Baines, R.A. Kaiser, T. Sheiko, W.J. Craigen, J.D. Molkentin, Voltage-dependent Ruegg, J.M. Dumont, K. Besseghir, E. Elmer, The nonimmunosuppressive anion channels are dispensable for mitochondrial-dependent cell death, Nat. Cell cyclosporin analogs NIM811 and UNIL025 display nanomolar potencies on Biol. 9 (2007) 550–555. permeability transition in brain-derived mitochondria, J. Bioenerg. Biomembr. [60] A.W. Leung, P. Varanyuwatana, A.P. Halestrap, The mitochondrial phosphate 36 (2004) 407–413. carrier interacts with cyclophilin D and may play a key role in the permeability [32] T. Tiepolo, A. Angelin, E. Palma, P. Sabatelli, L. Merlini, L. Nicolosi, F. Finetti, P. transition, J. Biol. Chem. 283 (2008) 26312–26323. Braghetta, G. Vuagniaux, J.M. Dumont, C. Baldari, P. Bonaldo, P. Bernardi, The [61] A.W. Leung, A.P. Halestrap, Recent progress in elucidating the molecular cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle mechanism of the mitochondrial permeability transition pore, Biochim. Biophys. apoptosis and ultrastructural defects in Col6a1 myopathic mice, Br. J. Pharmacol. Acta 1777 (2008) 946–952. (2009). [62] B.H. Kang, J. Plescia, T. Dohi, J. Rosa, S.J. Doxsey, D.C. Altieri, Regulation of tumor [33] S. Daum, M. Schumann, S. Mathea, T. Aumuller, M.A. Balsley, S.L. Constant, B.F. de cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone Lacroix, F. Kruska, M. Braun, C. Schiene-Fischer, Isoform-specific inhibition of network, Cell 131 (2007) 257–270. cyclophilins, Biochemistry 48 (2009) 6268–6277. [63] M. Juhaszova, D.B. Zorov, S.H. Kim, S. Pepe, Q. Fu, K.W. Fishbein, B.D. Ziman, S. [34] H. Ke, Q. Huai, Crystal structures of cyclophilin and its partners, Front Biosci. 9 Wang, K. Ytrehus, C.L. Antos, E.N. Olson, S.J. Sollott, Glycogen synthase kinase- (2004) 2285–2296. 3beta mediates convergence of protection signaling to inhibit the mitochondrial [35] X.J. Wang, F.A. Etzkorn, Peptidyl-prolyl isomerase inhibitors, Biopolymers 84 permeability transition pore, J. Clin. Invest. 113 (2004) 1535–1549. (2006) 125–146. [64] A. Rasola, M. Sciacovelli, F. Chiara, B. Pantic, W.S. Brusilow, P. Bernardi, Activation [36] N. Fournier, G. Ducet, A. Crevat, Action of cyclosporine on mitochondrial calcium of mitochondrial ERK protects cancer cells from death through inhibition of fluxes, J. Bioenerg. Biomembr. 19 (1987) 297–303. the permeability transition, Proc. Natl. Acad. Sci. USA 107 (2010) 726–731. [37] M. Crompton, H. Ellinger, A. Costi, Inhibition by cyclosporin A of a Ca2+-dependent [65] R.A. Eliseev, J. Malecki, T. Lester, Y. Zhang, J. Humphrey, T.E. Gunter, Cyclophilin D pore in heart mitochondria activated by inorganic phosphate and oxidative stress, interacts with Bcl2 and exerts an anti-apoptotic effect, J. Biol. Chem. 284 (2009) Biochem. J. 255 (1988) 357–360. 9692–9699. [38] K.M. Broekemeier, M.E. Dempsey, D.R. Pfeiffer, Cyclosporin A is a potent inhibitor [66] D.T. Lin, J.D. Lechleiter, Mitochondrial targeted cyclophilin D protects cells of the inner membrane permeability transition in liver mitochondria, J. Biol. Chem. from cell death by peptidyl prolyl isomerization, J. Biol. Chem. 277 (2002) 264 (1989) 7826–7830. 31134–31141. [39] A.P. Halestrap, A.M. Davidson, Inhibition of Ca2+-induced large-amplitude [67] V.K. Dickson, J.A. Silvester, I.M. Fearnley, A.G. Leslie, J.E. Walker, On the structure of swelling of liver and heart mitochondria by cyclosporin is probably caused by the stator of the mitochondrial ATP synthase, EMBO J. 25 (2006) 2911–2918. the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis– trans isomerase [68] I. Wittig, H. Schägger, Structural organization of mitochondrial ATP synthase, and preventing it interacting with the adenine nucleotide translocase, Biochem. J. Biochim. Biophys. Acta 1777 (2008) 592–598. 268 (1990) 153–160. [69] V. Giorgio, E. Bisetto, M.E. Soriano, F. Dabbeni-Sala, E. Basso, V. Petronilli, M.A. [40] I. Szabó, M. Zoratti, The giant channel of the inner mitochondrial membrane is Forte, P. Bernardi, G. Lippe, Cyclophilin D modulates mitochondrial FOF1 inhibited by cyclosporin A, J. Biol. Chem. 266 (1991) 3376–3379. ATP synthase by interacting with the lateral stalk of the complex, J. Biol. Chem. [41] M. Forte, P. Bernardi, Genetic dissection of the permeability transition pore, 284 (2009) 33982–33988. J. Bioenerg. Biomembr. 37 (2005) 121–128. [70] N.S. Green, E. Reisler, K.N. Houk, Quantitative evaluation of the lengths of [42] P. Bernardi, A. Krauskopf, E. Basso, V. Petronilli, E. Blachly-Dyson, F. Di Lisa, M.A. homobifunctional protein cross-linking reagents used as molecular rulers, Protein Forte, The mitochondrial permeability transition from in vitro artifact to disease Sci. 10 (2001) 1293–1304. target, FEBS J. 273 (2006) 2077–2099. [71] G.I. Belogrudov, J.M. Tomich, Y. Hatefi, ATP synthase complex. Proximities [43] A. Rasola, P. Bernardi, The mitochondrial permeability transition pore and its of subunits in bovine submitochondrial particles, J. Biol. Chem. 270 (1995) 2053–2060. involvement in cell death and in disease pathogenesis, Apoptosis 12 (2007) [72] M. Strauss, G. Hofhaus, R.R. Schroder, W. Kuhlbrandt, Dimer ribbons of ATP synthase 815–833. shape the inner mitochondrial membrane, EMBO J. 27 (2008) 1154–1160. [44] C.P. Baines, The molecular composition of the mitochondrial permeability [73] M. Campanella, E. Casswell, S. Chong, Z. Farah, M.R. Wieckowski, A.Y. Abramov, A. transition pore, J. Mol. Cell Cardiol. 46 (2009) 850–857. Tinker, M.R. Duchen, Regulation of mitochondrial structure and function by the [45] F. Di Lisa, P. Bernardi, A CaPful of mechanisms regulating the mitochondrial F1FO-ATPase inhibitor protein, IF1, Cell Metab. 8 (2008) 13–25. permeability transition, J. Mol. Cell. Cardiol. 46 (2009) 775–780. [74] D. Thomas, P. Bron, T. Weimann, A. Dautant, M.F. Giraud, P. Paumard, B. Salin, A. [46] C.P. Baines, R.A. Kaiser, N.H. Purcell, N.S. Blair, H. Osinska, M.A. Hambleton, E.W. Cavalier, J. Velours, D. Brethes, Supramolecular organization of the yeast F1FO-ATP Brunskill, M.R. Sayen, R.A. Gottlieb, G.W. Dorn, J. Robbins, J.D. Molkentin, Loss of synthase, Biol. Cell 100 (2008) 591–601. cyclophilin D reveals a critical role for mitochondrial permeability transition in [75] X. Wang, Y. Carlsson, E. Basso, C. Zhu, C.I. Rousset, A. Rasola, B.R. Johansson, K. cell death, Nature 434 (2005) 658–662. Blomgren, C. Mallard, P. Bernardi, M.A. Forte, H. Hagberg, Developmental shift of [47] E. Basso, L. Fante, J. Fowlkes, V. Petronilli, M.A. Forte, P. Bernardi, Properties of the cyclophilin D contribution to hypoxic–ischemic brain injury, J. Neurosci. 29 (2009) permeability transition pore in mitochondria devoid of Cyclophilin D, J. Biol. 2588–2596. Chem. 280 (2005) 18558–18561. [76] S. Luvisetto, E. Basso, V. Petronilli, P. Bernardi, M. Forte, Enhancement of anxiety, [48] T. Nakagawa, S. Shimizu, T. Watanabe, O. Yamaguchi, K. Otsu, H. Yamagata, H. facilitation of avoidance behavior, and occurrence of adult-onset obesity in mice Inohara, T. Kubo, Y. Tsujimoto, Cyclophilin D-dependent mitochondrial perme- lacking mitochondrial cyclophilin D, Neuroscience 155 (2008) 585–596. ability transition regulates some necrotic but not apoptotic cell death, Nature 434 [77] P.C. Waldmeier, J.J. Feldtrauer, T. Qian, J.J. Lemasters, Inhibition of the mitochondrial (2005) 652–658. permeability transition by the nonimmunosuppressive cyclosporin derivative [49] A.C. Schinzel, O. Takeuchi, Z. Huang, J.K. Fisher, Z. Zhou, J. Rubens, C. Hetz, N.N. NIM811, Mol. Pharmacol. 62 (2002) 22–29. Danial, M.A. Moskowitz, S.J. Korsmeyer, Cyclophilin D is a component of [78] E.J. Griffiths, A.P. Halestrap, Protection by Cyclosporin A of ischemia/reperfusion- mitochondrial permeability transition and mediates neuronal cell death after induced damage in isolated rat hearts, J. Mol. Cell. Cardiol. 25 (1993) 1461–1469. focal cerebral ischemia, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 12005–12010. [79] C. Piot, P. Croisille, P. Staat, H. Thibault, G. Rioufol, N. Mewton, R. Elbelghiti, T.T. [50] E. Basso, V. Petronilli, M.A. Forte, P. Bernardi, Phosphate is essential for inhibition Cung, E. Bonnefoy, D. Angoulvant, C. Macia, F. Raczka, C. Sportouch, G. Gahide, G. of the mitochondrial permeability transition pore by cyclosporin A and by Finet, X. André-Fouët, D. Revel, G. Kirkorian, J.-P. Monassier, G. Derumeaux, M. cyclophilin D ablation, J. Biol. Chem. 283 (2008) 26307–26311. Ovize, Effect of cyclosporine on reperfusion injury in acute myocardial infarction, [51] T.E. Gunter, D.R. Pfeiffer, Mechanisms by which mitochondria transport calcium, N. Engl. J. Med. 359 (2008) 473–481. Am. J. Physiol. 258 (1990) C755–C786. [80] H. Friberg, M. Ferrand-Drake, F. Bengtsson, A.P. Halestrap, T. Wieloch, Cyclosporin [52] K. Woodfield, A. Ruck, D. Brdiczka, A.P. Halestrap, Direct demonstration of a A, but not FK 506, protects mitochondria and neurons against hypoglycemic specific interaction between cyclophilin-D and the adenine nucleotide translocase damage and implicates the mitochondrial permeability transition in cell death, confirms their role in the mitochondrial permeability transition, Biochem. J. 336 J. Neurosci. 18 (1998) 5151–5159. (Pt 2) (1998) 287–290. [81] J. Folbergrova, P.A. Li, H. Uchino, M.L. Smith, B.K. Siesjo, Changes in the [53] A.P. Halestrap, C. Brenner, The adenine nucleotide translocase: a central bioenergetic state of rat hippocampus during 2.5 min of ischemia, and prevention component of the mitochondrial permeability transition pore and key player in of cell damage by cyclosporin A in hyperglycemic subjects, Exp. Brain Res. 114 cell death, Curr. Med. Chem. 10 (2003) 1507–1525. (1997) 44–50. [54] M. Crompton, S. Virji, J.M. Ward, Cyclophilin-D binds strongly to complexes of the [82] H. Uchino, E. Elmer, K. Uchino, P.A. Li, Q.P. He, M.L. Smith, B.K. Siesjo, Amelioration voltage-dependent anion channel and the adenine nucleotide translocase to form by cyclosporin A of brain damage in transient forebrain ischemia in the rat, Brain the permeability transition pore, Eur. J. Biochem. 258 (1998) 729–735. Res. 812 (1998) 216–226. 1118 V. Giorgio et al. / Biochimica et Biophysica Acta 1797 (2010) 1113–1118

[83] S. Matsumoto, H. Friberg, M. Ferrand-Drake, T. Wieloch, Blockade of the [92] O. Camacho Vanegas, E. Bertini, R.Z. Zhang, S. Petrini, C. Minosse, P. Sabatelli, B. Giusti, mitochondrial permeability transition pore diminishes infarct size in the rat M.L. Chu, G. Pepe, Ullrich scleroatonic muscular dystrophy is caused by recessive after transient middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 19 mutations in collagen type VI, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 7516–7521. (1999) 736–741. [93] L. Merlini, E. Martoni, P. Grumati, P. Sabatelli, S. Squarzoni, A. Urciuolo, A. Ferlini, F. [84] D.O. Okonkwo, J.T. Povlishock, An intrathecal bolus of cyclosporin A before injury Gualandi, P. Bonaldo, Autosomal recessive myosclerosis myopathy is a collagen VI preserves mitochondrial integrity and attenuates axonal disruption in traumatic disorder, Neurology 71 (2008) 1245–1253. brain injury, J. Cereb. Blood Flow Metab. 19 (1999) 443–451. [94] P. Bonaldo, P. Braghetta, M. Zanetti, S. Piccolo, D. Volpin, G.M. Bressan, Collagen VI [85] S.W. Scheff, P.G. Sullivan, Cyclosporin A significantly ameliorates cortical damage deficiency induces early onset myopathy in the mouse: an animal model for following experimental traumatic brain injury in rodents, J. Neurotrauma 16 Bethlem myopathy, Hum. Mol. Genet. 7 (1998) 2135–2140. (1999) 783–792. [95] W.A. Irwin, N. Bergamin, P. Sabatelli, C. Reggiani, A. Megighian, L. Merlini, P. [86] M. Forte, B.G. Gold, G. Marracci, P. Chaudhary, E. Basso, D. Johnsen, X. Yu, J. Braghetta, M. Columbaro, D. Volpin, G.M. Bressan, P. Bernardi, P. Bonaldo, Fowlkes, M. Rahder, K. Stem, P. Bernardi, D. Bourdette, Cyclophilin D inactivation Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI protects axons in experimental autoimmune encephalomyelitis, an animal model deficiency, Nat. Genet. 35 (2003) 267–271. of multiple sclerosis, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 7558–7563. [96] E. Palma, T. Tiepolo, A. Angelin, P. Sabatelli, N.M. Maraldi, E. Basso, M.A. Forte, P. [87] L.J. Martin, B. Gertz, Y. Pan, A.C. Price, J.D. Molkentin, Q. Chang, The mitochondrial Bernardi, P. Bonaldo, Genetic ablation of cyclophilin D rescues mitochondrial permeability transition pore in motor neurons: involvement in the pathobiology defects and prevents muscle apoptosis in collagen VI myopathic mice, Hum. Mol. of ALS mice, Exp. Neurol. 218 (2009) 333–346. Genet. 18 (2009) 2024–2031. [88] H. Du, L. Guo, F. Fang, D. Chen, A.A. Sosunov, G.M. McKhann, Y. Yan, C. Wang, H. [97] A. Angelin, T. Tiepolo, P. Sabatelli, P. Grumati, N. Bergamin, C. Golfieri, E. Mattioli, F. Zhang, J.D. Molkentin, F.J. Gunn-Moore, J.P. Vonsattel, O. Arancio, J.X. Chen, S.D. Gualandi, A. Ferlini, L. Merlini, N.M. Maraldi, P. Bonaldo, P. Bernardi, Mitochondrial Yan, Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation dysfunction in the pathogenesis of Ullrich congenital muscular dystrophy and and ameliorates learning and memory in Alzheimer's disease, Nat. Med. 14 (2008) prospective therapy with cyclosporins, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 1097–1105. 991–996. [89] A.K. Lampe, K.M. Bushby, Collagen VI related muscle disorders, J. Med. Genet. 42 [98] L. Merlini, A. Angelin, T. Tiepolo, P. Braghetta, P. Sabatelli, A. Zamparelli, A. Ferlini, (2005) 673–685. N.M. Maraldi, P. Bonaldo, P. Bernardi, Cyclosporin A corrects mitochondrial [90] G.J. Jöbsis, H. Keizers, J.P. Vreijling, M. de Visser, M.C. Speer, R.A. Wolterman, F. dysfunction and muscle apoptosis in patients with collagen VI myopathies, Proc. Baas, P.A. Bolhuis, Type VI collagen mutations in Bethlem myopathy, an autosomal Natl. Acad. Sci. U. S. A. 105 (2008) 5225–5229. dominant myopathy with contractures, Nat. Genet. 14 (1996) 113–115. [99] D.P. Millay, M.A. Sargent, H. Osinska, C.P. Baines, E.R. Barton, G. Vuagniaux, H.L. [91] O. Ullrich, Kongenitale, atonisch-sklerotische Muskeldystrophie, ein weiterer Typus Sweeney, J. Robbins, J.D. Molkentin, Genetic and pharmacologic inhibition of der heredodegenerativen Erkrankungen des neuromuskulaeren Systems, Z. Ges. mitochondrial-dependent necrosis attenuates muscular dystrophy, Nat. Med. 14 Neurol. Psychiatr. 126 (1930) 171–201. (2008) 442–447.