Mitochondrial dynamics in cell life and death

Mitochondria are central organelles for the life and death of the cell. They provide most of the ATP required for endoergonic processes, participate in crucial biosynthetic pathways, shape Ca²⁺ signalling and regulate cell death. The functional versatility of mitochondria is paralleled by their morphological complexity. In certain cell types mitochondria are organized in networks of interconnected organelles. Ultrastructurally, the inner membrane (IM) can be further subdivided in an inner boundary membrane and in the cristae compartment, bag-like folds of the IM connected to it via narrow tubular junctions. "Mitochondria-shaping" proteins impinge on the equilibrium between fusion and fission, which ultimately determines the ultrastructural and cellular morphology of the organelle. Changes in mitochondrial shape appear to regulate crucial mitochondrial and cellular functions. Our initial contribution changed the general consensus that mitochondria remain untouched during apoptosis, when we described that during programmed cell death they remodel their inner structure to allow the bulk of cytochrome c to be released from the cristae stores. Since then, we use an integrated approach of genetics, advanced imaging, biochemistry, physiology and electron tomography to unravel the role of mitochondria-shaping in cell life and death.

Molecular mechanisms of OPA1 functions

Figure 1. Cartoon representing the role of OPA1 oligomerization in keeping the cristae in shape and in the regulation of cytochrome c release during apoptosis. Artwork: Eric Smith, Boston.

Figure 1. Cartoon representing the role of OPA1 oligomerization in keeping the cristae in shape and in the regulation of cytochrome c release during apoptosis. Artwork: Eric Smith, Boston.
[click image to enlarge]

We have shown that the inner mitochondrial membrane dynamin-related protein Optic atrophy 1 (Opa1) has multiple, genetically distinguishable functions in mitochondrial fusion and in the control of cristae remodelling and cytochrome c release. While the pro-fusion effect depends on the outer membrane protein of the same family Mitofusin 1 (Cipolat et al, PNAS, 2004), the remodelling of the cristae depends on the inner mitochondrial membrane rhomboid protease Parl (Cipolat et al., Cell, 2006). This is required for the complete processing of Opa1 into a soluble, intermembrane space form that participates together with the membrane bound one in the formation of a high molecular weight oligomer. Opa1-containing oligomers are early targets during remodelling of the cristae in the course of apoptosis (Frezza et al., Cell, 2006). We now plan to extend our analysis to understand at the molecular mechanism the function of Opa1 in these two genetically distinguishable pathways. We capitalize on mouse models of Opa1 overexpression and conditional ablation to identify the partners of Opa1 in these high-molecular weight complexes in normal and apoptotic mitochondria and to identify their relative role in one of the two processes controlled by Opa1. These mouse models are also instrumental to elucidate the role of Opa1 in retinal ganglion cells, which are selectively affected in Dominant Optic Atrophy, caused by mutations in Opa1. Moreover, we wish to pharmacologically target Opa1 to verify if this can augment the susceptibility to antineoplastic drugs.

Molecular anatomy and pathophysiology of the endoplasmic reticulum-mitochondria interface

Figure 2. representative images of mitochondria (red)-ER (green) tethering (yellow) in wt (A) and Mfn2-/- (B) mouse embryonic fibroblasts.

Figure 2. representative images of mitochondria (red)-ER (green) tethering (yellow) in wt (A) and Mfn2-/- (B) mouse embryonic fibroblasts.
[click image to enlarge]

Organelles are not randomly organized in the cytoplasm of the cell, but often are orderly arranged in mutual relationships that depend on physical, protein bounds. Understanding the molecular nature of the tethers that regulate relative position and juxtaposition of the organelles is one of the main quests of cell biology, given their functional importance. For example, the juxtaposition between mitochondria and endoplasmic reticulum (ER) has been suggested by us and others to crucially impact on Ca²⁺ signalling and apoptosis. We recently identified the first structural ER-mitochondrial tether in mitofusin 2 (Mfn2), a pro-fusion mitochondria-shaping protein. A fraction of Mfn2 is also located on the ER regulating its morphology, and acting in trans to tether it to mitochondria (de Brito and Scorrano, Nature, 2008). The tethering function of Mfn2 impacts on the transmission of Ca²⁺ signals between the two organelles and is regulated by the oncosuppressor trichoplein/mitostatin (Cerqua et al. EMBO Rep. 2010). Mfn2 is likely only one of the tethers, as others exist in yeast. Furthermore, the dynamicity of the ER-mitochondria contact is known, but remains poorly understood. Therefore, a clear picture of the anatomy and pathophsyiology of ER-mitochondrial connection is far from being reached. We are exploring the possibility that ER-mitochondrial contacts are crucial specialized hubs of cellular signalling whose architecture is modulated by cellular cues, impacting on integrated signalling cascades and ultimately affecting cellular function. To this end we are integrating genomics, proteomics, imaging and physiology to discover the molecular nature of tethers and modulators of ER-mitochondrial tethers in mammalian cells; clarify how mitochondrial and ER function are controlled by the tethering; address how juxtaposition influences complex cellular responses including autophagy and cell death; elucidate the role of tethering in vivo by generating animal models with defined ER-mitochondrial distance.

Mitochondrial shape changes and autophagy

We are using a genetic approach to investigate whether fission and/or dysfunction are required to induce elimination of the organelle by autophagy, a bulk cellular recycling and degradation process. Our data indicate that a signaling cascade impinges on mitochondrial morphology to determine the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in cultured cells as well as in the whole animal. Upon starvation, a powerful inducer of autophagy, cellular cAMP levels increase and protein kinase A (PKA) becomes activated. PKA in turn phosphorylates the pro-fission dynamin related protein 1 (Drp1) that is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increase dimerization and activity of ATP synthase, and maintain ATP production. When elongation is genetically or pharmacologically blocked, mitochondria conversely consume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology participate in the decision of the fate of the cell during autophagy. We are currently extending our findings to address how dysfunctional mitochondria communicate with the autophagy machinery.

The role of mitochondrial dynamics in Huntington's Disease

Huntington's Disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Our data indicate that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells the increased basal activity of the phosphatase calcineurin dephosphorylates the pro-fission Drp1, increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis (Costa et al., EMBO Mol. Med., 2010). We are extending our results by moving to animal models of HD where Drp1 is selectively ablated in the striatum, to clarify if changes in mitochondrial morphology play any role in the natural history of HD.

Synoptic CV

Honours

Selected VIMM Publications

• Costa V, Giacomello M, Hudec R, Lopreiato R, Ermak G, Lim D, Malorni W, Davies KJ, Carafoli E, Scorrano L (2010) Mitochondrial fission and cristae disruption increase the response of cell models of Huntington's disease to apoptotic stimuli. EMBO Mol Med 2:490-503.
• Cerqua C, Anesti V, Pyakurel A, Liu D, Naon D, Wiche G, Baffa R, Dimmer KS, Scorrano L (2010) Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition. EMBO Rep. 11:854-60.
• de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456:605-10.
• Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, Scorrano L (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126:177-89.
• Cipolat S, Rudka T, Hartmann D, Costa V, Serneels L, Craessaerts K, Metzger K, Frezza C, Annaert W, D'Adamio L, Derks C, Dejaegere T, Pellegrini L, D'Hooge R, Scorrano L, De Strooper B (2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126:163-75.

VIMM Publications

• de Brito OM, Scorrano L (2010) An intimate liaison: spatial organization of the endoplasmic reticulum-mitochondria relationship. EMBO J. 29:2715-23.
• Cereghetti GM, Costa V, Scorrano L (2010) Inhibition of Drp1-dependent mitochondrial fragmentation and apoptosis by a polypeptide antagonist of calcineurin. Cell Death Differ. 17:1785-94.
• Morais VA, Verstreken P, Roethig A, Smet J, Snellinx A, Vanbrabant M, Haddad D, Frezza C, Mandemakers W, Vogt-Weisenhorn D, Van Coster R, Wurst W, Scorrano L, De Strooper B (2009) Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function. EMBO Mol Med 1:99-111.
• Wasilewski M, Scorrano L (2009) The changing shape of mitochondrial apoptosis. Trends Endocrinol. Metab. 20:287-94.
• Demaurex N, Scorrano L (2009) Reactive oxygen species are NOXious for neurons. Nat. Neurosci. 12:819-20.
• Lamarca V, Scorrano L (2009) When separation means death: killing through the mitochondria, but starting from the endoplasmic reticulum. EMBO J. 28:1681-3.
• Scorrano L, Liu D (2009) The SUMO arena goes mitochondrial with MAPL. EMBO Rep. 10:694-6.
• Scorrano L (2009) Opening the doors to cytochrome c: changes in mitochondrial shape and apoptosis. Int. J. Biochem. Cell Biol. 41:1875-83.
• Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, Bazan NG, Blagosklonny MV, Blomgren K, Borner C, Bredesen DE, Brenner C, Castedo M, Cidlowski JA, Ciechanover A, Cohen GM, De Laurenzi V, De Maria R, Deshmukh M, Dynlacht BD, El-Deiry WS, Flavell RA, Fulda S, Garrido C, Golstein P, Gougeon ML, Green DR, Gronemeyer H, Hajnóczky G, Hardwick JM, Hengartner MO, Ichijo H, Jäättelä M, Kepp O, Kimchi A, Klionsky DJ, Knight RA, Kornbluth S, Kumar S, Levine B, Lipton SA, Lugli E, Madeo F, Malomi W, Marine JC, Martin SJ, Medema JP, Mehlen P, Melino G, Moll UM, Morselli E, Nagata S, Nicholson DW, Nicotera P, Nuñez G, Oren M, Penninger J, Pervaiz S, Peter ME, Piacentini M, Prehn JH, Puthalakath H, Rabinovich GA, Rizzuto R, Rodrigues CM, Rubinsztein DC, Rudel T, Scorrano L, Simon HU, Steller H, Tschopp J, Tsujimoto Y, Vandenabeele P, Vitale I, Vousden KH, Youle RJ, Yuan J, Zhivotovsky B, Kroemer G (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ. 16:1093-107.
• de Brito OM, Scorrano L (2009) Mitofusin-2 regulates mitochondrial and endoplasmic reticulum morphology and tethering: the role of Ras. Mitochondrion 9:222-6.
• Scorrano L (2008) Caspase-8 goes cardiolipin: a new platform to provide mitochondria with microdomains of apoptotic signals? J. Cell Biol. 183:579-81.
• Vecchione A, Fassan M, Anesti V, Morrione A, Goldoni S, Baldassarre G, Byrne D, D'Arca D, Palazzo JP, Lloyd J, Scorrano L, Gomella LG, Iozzo RV, Baffa R (2009) MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer. Oncogene 28:257-69.
• Cereghetti GM, Stangherlin A, Martins de Brito O, Chang CR, Blackstone C, Bernardi P, Scorrano L (2008) Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl. Acad. Sci. U.S.A. 105:15803-8.
• Spinazzi M, Cazzola S, Bortolozzi M, Baracca A, Loro E, Casarin A, Solaini G, Sgarbi G, Casalena G, Cenacchi G, Malena A, Frezza C, Carrara F, Angelini C, Scorrano L, Salviati L, Vergani L (2008) A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function. Hum. Mol. Genet. 17:3291-302.
• Gomes LC, Scorrano L (2008) High levels of Fis1, a pro-fission mitochondrial protein, trigger autophagy. Biochim. Biophys. Acta 1777:860-6.
• Frezza C, Cipolat S, Scorrano L (2007) Measuring mitochondrial shape changes and their consequences on mitochondrial involvement during apoptosis. Methods Mol. Biol. 372:405-20.
• de Brito OM, Scorrano L (2008) Mitofusin 2: a mitochondria-shaping protein with signaling roles beyond fusion. Antioxid. Redox Signal. 10:621-33.
• Scorrano L (2007) Multiple functions of mitochondria-shaping proteins. Novartis Found. Symp. 287:47-55; discussion 55.
• Dimmer KS, Navoni F, Casarin A, Trevisson E, Endele S, Winterpacht A, Salviati L, Scorrano L (2008) LETM1, deleted in Wolf-Hirschhorn syndrome is required for normal mitochondrial morphology and cellular viability. Hum. Mol. Genet. 17:201-14.
• Hausenloy DJ, Scorrano L (2007) Targeting cell death. Clin. Pharmacol. Ther. 82:370-3.
• Pellegrini L, Scorrano L (2007) A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis. Cell Death Differ. 14:1275-84.
• Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287-95.
• Campello S, Lacalle RA, Bettella M, Mañes S, Scorrano L, Viola A (2006) Orchestration of lymphocyte chemotaxis by mitochondrial dynamics. J. Exp. Med. 203:2879-86.
• Alirol E, James D, Huber D, Marchetto A, Vergani L, Martinou JC, Scorrano L (2006) The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis. Mol. Biol. Cell 17:4593-605.
• Cereghetti GM, Scorrano L (2006) The many shapes of mitochondrial death. Oncogene 25:4717-24.
• Dimmer KS, Scorrano L (2006) (De)constructing mitochondria: what for? 21:233-41.
• Cipolat S, Scorrano L (2006) To fuse and to protect. A novel role for CED-9 in mitochondrial morphology reveals an ancient function. Cell Death Differ. 13:1833-4.
• Letai A, Scorrano L (2006) Laying the foundations of programmed cell death. Cell Death Differ. 13:1245-7.
• Anesti V, Scorrano L (2006) The relationship between mitochondrial shape and function and the cytoskeleton. Biochim. Biophys. Acta 1757:692-9.
• Buytaert E, Callewaert G, Hendrickx N, Scorrano L, Hartmann D, Missiaen L, Vandenheede JR, Heirman I, Grooten J, Agostinis P (2006) Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy. FASEB J. 20:756-8.
• Scorrano L (2005) Proteins that fuse and fragment mitochondria in apoptosis: con-fissing a deadly con-fusion? J. Bioenerg. Biomembr. 37:165-70.
• Scorrano L (2005) Stanley J. Korsmeyer (1950-2005). J. Bioenerg. Biomembr. 37:109.
• Cipolat S, Martins de Brito O, Dal Zilio B, Scorrano L (2004) OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl. Acad. Sci. U.S.A. 101:15927-32.

Additional Publications

• Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD, Pozzan T, Korsmeyer SJ (2003) BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300:135-9.
• Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA, Korsmeyer SJ (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev. Cell 2:55-67.

Selected Seminars

Contact

Luca Scorrano Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 221 Fax:  (+39) 049 7923 271