Paolo Bernardi
Michael Forte- 1Department of Biomedical Sciences, University of Padova, Padova, Italy
- 2Vollum Institute, Oregon Health and Science University, Portland, OR, USA
A commentary on
SPG7 is an essential and conserved component of the mitochondrial permeability transition pore.
by Shanmughapriya, S., Rajan, S., Hoffman, N. E., Higgins, A. M., Tomar, D., Nemani, N., et al. (2015). Mol. Cell 60, 47–62. doi: 10.1016/j.molcel.2015.08.009
SPG7 (paraplegin) is the product of the SPG7 gene, whose mutations are responsible for an autosomal recessive form of hereditary spastic paraplegia (HSP) (De Michele et al., 1998). SPG7 is a AAA-protease (Casari et al., 1998) that co-assembles with a homologous protein, AFG3L, in the inner mitochondrial membrane. These proteins associate with unidentified proteins in high molecular weight complexes of up to 900 kDa, which are aberrant in HSP patient cells (Atorino et al., 2003; Koppen et al., 2007). Loss of this complex following deletion of SPG7 causes decreased activity of respiratory complex I and increased sensitivity to reactive oxygen species (ROS); both events can be rescued by expression of SPG7 (Atorino et al., 2003). A recent paper suggests that SPG7 also serves an essential role in the formation and regulation of the mitochondrial permeability transition pore (PTP) (Shanmughapriya et al., 2015).
The PTP is an inner membrane channel that forms after a permissive load of matrix Ca2+ under conditions of oxidative stress (Bernardi, 2013; Bernardi et al., 2015). Strong evidence indicates that it derives from the F-ATP synthase, which forms channels with electrophysiological features matching those expected of the PTP in mammals (Giorgio et al., 2013; Alavian et al., 2014), yeast (Carraro et al., 2014), and Drosophila (von Stockum et al., 2015). To identify regulators of the PTP, Shanmughapriya et al. used a phenotypic screen based on the mitochondrial Ca2+ retention capacity (CRC) of digitonin-permeabilized cultured human cells after treatment with siRNAs designed to suppress translation of a set of mitochondrial proteins (Shanmughapriya et al., 2015). This assay (Murphy et al., 1996; Fontaine et al., 1998) is based on the assumption that the CRC reflects the state of the PTP in situ, i.e. its propensity to open after treatment with Ca2+ and PTP agonists. The screen identified 13 proteins whose suppression caused desensitization of the PTP to Ca2+ with an increase of the CRC. The hits included well-known modulators that do not take part in PTP formation like cyclophilin (CyP) D, the matrix receptor that mediates the PTP inhibitory effect of cyclosporin A. The Authors identified SPG7 amongst the hits, and selected it for further study because it could be co-immunoprecipitated with CyPD in a complex that also included outer membrane VDAC1 (Shanmughapriya et al., 2015). Elimination of SPG7 expression by Cas9/CRISPR methods conferred protection from Ca2+- and oxidant-induced PTP opening and from cell death, as expected based on PTP desensitization. The Authors conclude that SPG7 is an essential component of the PTP complex together with VDAC1, but from analysis of the results we must conclude that this is an overinterpretation that is not supported by the experimental results presented.
First and foremost, the phenotypic screen does not allow a distinction between core PTP components from regulators. This difference–only core component of the PTP must necessarily be essential to PTP formation while regulators may only modulate PTP activity- represents important and mechanistically discrete phenomena. Indeed, failure to appreciate this critical difference has often confused our understanding of the molecular composition of the PTP. In this study, the results show that the PTP opened, albeit at higher Ca2+ loads, after suppression of all 13 transcripts including SPG7 (Shanmughapriya et al., 2015). Thus, the PTP opens even in the absence of SPG7, much as it does in the absence of CyPD (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005) questioning the conclusion that the protein is an essential component of the pore. Second, the mammalian PTP displays conductances up to 1.2 nS (Szabo and Zoratti, 2014) that are unlikely to be generated by the 2-transmembrane domain proteins SPG7 and AFG3L. Indeed, and in spite of its claims, the study of Shanmughapriya et al. does not address the question of whether the putative “PTP complex” formed by SPG7, AFG3L, and VDAC1 can actually form channels at all. Thus, the graphical abstract depicting the PTP as a complex of SPG7, AFG3L, and VDAC1 is a misrepresentation of the actual findings of the paper and of the literature on the PTP. The putative role of VDAC1 deserves a specific comment.
VDAC1 is the major outer membrane protein and one of the most abundant mitochondrial proteins in mammals. Its association with the PTP was suggested based on co-purification with other putative components, i.e. the adenine nucleotide translocase and the peripheral benzodiazepine receptor, today called TSPO (McEnery et al., 1992). The link was made because ligands of TSPO are also agonists of the PTP (Kinnally et al., 1993). As shown by experiments on mitochondria from mice where the corresponding genes were deleted, neither TSPO (Šileikyte et al., 2014) nor VDAC1 (Krauskopf et al., 2006) is an essential component of the PTP or a regulator of its activity, and the effects of “TSPO ligands” on the pore could rather be explained by their interaction with the F-ATP synthase (Cleary et al., 2007; Giorgio et al., 2013). Of note, also genetic inactivation of the less abundant VDAC2 and VDAC3 isoforms does not affect PTP opening and PTP-dependent cell death (Baines et al., 2007). Thus, the reported co-immunoprecipitation of SPG7 with VDAC1 (Shanmughapriya et al., 2015) does not bear on the nature or regulation of the PTP. Our comment does not imply that the outer mitochondrial membrane does not regulate PTP activity, as discussed in detail (Bernardi et al., 2015).
Surprisingly, Shanmughapriya et al. do not discuss possible mechanisms through which SPG7 may regulate the PTP. We suspect that the high molecular weight complex formed by SPG7 and AFG3L reported by Casari and coworkers (Atorino et al., 2003; Koppen et al., 2007) may be due to a direct interaction of the AAA-protease heterodimers with F-ATP synthase, which may in turn stabilize dimers/oligomers of the complex and thus favor Ca2+-dependent PTP formation (Bernardi et al., 2015). Increased oxidative stress due to inhibition of complex I could easily explain sensitization of the PTP. Thus, SPG7 could be one more of the many regulators of the PTP, but not an essential component of the pore.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
Research in our laboratories is supported by Telethon (GGP14037 and GPP14187), AIRC (IG13392), Ministry of the University and Research, Italy and NIH-PHS, USA (R01GM069883 and 03DA033978-01).
References
Alavian, K. N., Beutner, G., Lazrove, E., Sacchetti, S., Park, H. A., Licznerski, P., et al. (2014). An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore. Proc. Natl. Acad. Sci. U.S.A. 111, 10580–10585. doi: 10.1073/pnas.1401591111
Atorino, L., Silvestri, L., Koppen, M., Cassina, L., Ballabio, A., Marconi, R., et al. (2003). Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J. Cell Biol. 163, 777–787. doi: 10.1083/jcb.200304112
Baines, C. P., Kaiser, R. A., Purcell, N. H., Blair, N. S., Osinska, H., Hambleton, M. A., et al. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658–662. doi: 10.1038/nature03434
Baines, C. P., Kaiser, R. A., Sheiko, T., Craigen, W. J., and Molkentin, J. D. (2007). Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat. Cell Biol. 9, 550–555. doi: 10.1038/ncb1575
Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Forte, M. A., and Bernardi, P. (2005). Properties of the permeability transition pore in mitochondria devoid of cyclophilin D. J. Biol. Chem. 280, 18558–18561. doi: 10.1074/jbc.C500089200
Bernardi, P. (2013). The mitochondrial permeability transition pore: a mystery solved? Front. Physiol. 4:95. doi: 10.1016/b978-0-12-378630-2.00151-1
Bernardi, P., Rasola, A., Forte, M., and Lippe, G. (2015). The Mitochondrial permeability transition pore: channel formation by F-ATP synthase, integration in signal transduction, and role in pathophysiology. Physiol. Rev. 95, 1111–1155. doi: 10.1152/physrev.00001.2015
Carraro, M., Giorgio, V., Šileikyte, J., Sartori, G., Forte, M., Lippe, G., et al. (2014). Channel formation by Yeast F-ATP synthase and the role of dimerization in the mitochondrial permeability transition. J. Biol. Chem. 289, 15980–15985. doi: 10.1074/jbc.C114.559633
Casari, G., De Fusco, M., Ciarmatori, S., Zeviani, M., Mora, M., Fernandez, P., et al. (1998). Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93, 973–983. doi: 10.1016/S0092-8674(00)81203-9
Cleary, J., Johnson, K. M., Opipari, A. W. Jr., and Glick, G. D. (2007). Inhibition of the mitochondrial F1F0-ATPase by ligands of the peripheral benzodiazepine receptor. Bioorg. Med Chem. Lett. 17, 1667–1670. doi: 10.1016/j.bmcl.2006.12.102
De Michele, G., De Fusco, M., Cavalcanti, F., Filla, A., Marconi, R., Volpe, G., et al. (1998). A new locus for autosomal recessive hereditary spastic paraplegia maps to chromosome 16q24.3. Am. J. Hum. Genet. 63, 135–139. doi: 10.1086/301930
Fontaine, E., Ichas, F., and Bernardi, P. (1998). A ubiquinone-binding site regulates the mitochondrial permeability transition pore. J. Biol. Chem. 273, 25734–25740. doi: 10.1074/jbc.273.40.25734
Giorgio, V., von Stockum, S., Antoniel, M., Fabbro, A., Fogolari, F., Forte, M., et al. (2013). Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc. Natl. Acad. Sci. U.S.A. 110, 5887–5892. doi: 10.1073/pnas.1217823110
Kinnally, K. W., Zorov, D. B., Antonenko, Y. N., Snyder, S. H., McEnery, M. W., and Tedeschi, H. (1993). Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands. Proc. Natl. Acad. Sci. U.S.A. 90, 1374–1378. doi: 10.1073/pnas.90.4.1374
Koppen, M., Metodiev, M. D., Casari, G., Rugarli, E. I., and Langer, T. (2007). Variable and tissue-specific subunit composition of mitochondrial m-AAA protease complexes linked to hereditary spastic paraplegia. Mol. Cell Biol. 27, 758–767. doi: 10.1128/MCB.01470-06
Krauskopf, A., Eriksson, O., Craigen, W. J., Forte, M. A., and Bernardi, P. (2006). Properties of the permeability transition in VDAC1−/− mitochondria. Biochim. Biophys. Acta 1757, 590–595. doi: 10.1016/j.bbabio.2006.02.007
McEnery, M. W., Snowman, A. M., Trifiletti, R. R., and Snyder, S. H. (1992). Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc. Natl. Acad. Sci. U.S.A. 89, 3170–3174. doi: 10.1073/pnas.89.8.3170
Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., and Fiskum, G. (1996). Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. U.S.A. 93, 9893–9898. doi: 10.1073/pnas.93.18.9893
Nakagawa, T., Shimizu, S., Watanabe, T., Yamaguchi, O., Otsu, K., Yamagata, H., et al. (2005). Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 434, 652–658. doi: 10.1038/nature03317
Schinzel, A. C., Takeuchi, O., Huang, Z., Fisher, J. K., Zhou, Z., Rubens, J., et al. (2005). Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc. Natl. Acad. Sci. U.S.A. 102, 12005–12010. doi: 10.1073/pnas.0505294102
Shanmughapriya, S., Rajan, S., Hoffman, N. E., Higgins, A. M., Tomar, D., Nemani, N., et al. (2015). SPG7 is an essential and conserved component of the mitochondrial permeability transition pore. Mol. Cell 60, 47–62. doi: 10.1016/j.molcel.2015.08.009
Šileikyte, J., Blachly-Dyson, E., Sewell, R., Carpi, A., Menabò, R., Di Lisa, F., et al. (2014). Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (translocator protein of 18 kDa (TSPO)). J. Biol. Chem. 289, 13769–13781. doi: 10.1074/jbc.M114.549634
Szabo, I., and Zoratti, M. (2014). Mitochondrial channels: ion fluxes and more. Physiol. Rev. 94, 519–608. doi: 10.1152/physrev.00021.2013
Keywords: mitochondria, permeability transition pore, SPG7, paraplegin, cell death
Citation: Bernardi P and Forte M (2015) Commentary: SPG7 is an essential and conserved component of the mitochondrial permeability transition pore. Front. Physiol. 6:320. doi: 10.3389/fphys.2015.00320
Received: 24 September 2015; Accepted: 22 October 2015;
Published: 04 November 2015.
Edited by:
Gyorgy Hajnoczky, Thomas Jefferson University, USAReviewed by:
Atan Gross, The Weizmann Institute of Science, IsraelJan B. Hoek, Thomas Jefferson University, USA
Copyright © 2015 Bernardi and Forte. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Paolo Bernardi, bernardi@bio.unipd.it;
Michael Forte, forte@ohsu.edu