Luca Scorrano

The Mitochondrial Permeability Transition, Release of Cytochrome c and Cell Death CORRELATION WITH THE DURATION OF PORE OPENINGS IN SITU

We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Treatment with arachidonic acid or A23187 induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co2+ quenching technique (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725–734). Yet depolarization, as assessed from the changes of mitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapid and extensive with arachidonic acid and slow and partial withA23187. Cyclosporin A-inhibitable release of cytochrome cand cell death correlate with the changes of TMRM fluorescence but not with those of calcein fluorescence. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. Modulation of the open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.

The many shapes of mitochondrial death

Mitochondria integrate apoptotic signalling by releasing cytochrome c and other proapoptotic cofactors needed for activation of effector caspases. Previously overlooked morphological changes, mitochondrial fragmentation and cristae remodelling, emerged as subroutines of the mitochondrial programme of apoptosis in mammalian cells, as well as in developmental cell death of Caenorhabditis elegans. Mitochondrial morphology results from fusion and fission processes, controlled by a growing set of ‘mitochondria-shaping’ proteins. Their levels and function appear to influence mitochondrial pathways of cell death, but mechanisms are largely unknown. An emerging model implicates different signals converging on mitochondria-shaping proteins to activate or deactivate them during apoptosis. In turn, these proteins can orchestrate changes in mitochondrial shape to insure cytochrome c release and progression of the apoptotic cascade. These therefore appear an appealing novel therapeutic target to modulate cell death in cancer.

The changing shape of mitochondrial apoptosis

Mitochondria are key organelles in conversion of energy, regulation of cellular signaling and amplification of programmed cell death. The anatomy of the organelle matches this functional versatility in complexity and is modulated by the concerted action of proteins that impinge on its fusion-fission equilibrium. A growing body of evidence implicates changes in mitochondrial shape in the progression of apoptosis and, therefore, proteins governing such changes are likely candidates for involvement in pathogenetic mechanisms in neurodegeneration and cancer. Here, we discuss the recent advancements in our knowledge about the machinery that regulates mitochondrial shape and on the role of molecular mechanisms controlling mitochondrial morphology during cell death.

Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition

Trichoplein/mitostatin (TpMs) is a keratin‐binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)‐dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca2+‐dependent stimuli that require ER–mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria–ER juxtaposition.

Two Close, Too Close: Sarcoplasmic Reticulum–Mitochondrial Crosstalk and Cardiomyocyte Fate

Mitochondria are key organelles in cell life whose dysfunction is associated with a variety of diseases. Their crucial role in intermediary metabolism and energy conversion makes them a preferred target in tissues, such as the heart, where the energetic demands are very high. In the cardiomyocyte, the spatial organization of mitochondria favors their interaction with the sarcoplasmic reticulum, thereby offering a mechanism for Ca(2+)-mediated crosstalk between these 2 organelles. Recently, the molecular basis for this interaction has begun to be unraveled, and we are learning how endoplasmic reticulum-mitochondrial interactions are often exploited by death signals, such as proapoptotic Bcl-2 family members, to amplify the cell death cascade. Here, we review our present understanding of the structural basis and the functional consequences of the close interaction between sarcoplasmic reticulum and mitochondria on cardiomyocyte function and death.

Two modes of activation of the permeability transition pore: The role of mitochondrial cyclophilin

Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CsA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-ds-rrafts-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M. (Mol Cell Biochem 174: 181–184,1997)

The Pathophysiology of LETM1

Originally identified as a key element of mitochondrial volume homeostasis through regulation of K+–H+ exchange (KHE), the LETM1 protein family is also involved in respiratory chain biogenesis and in the pathogenesis of seizures in the Wolf–Hirschhorn syndrome (WHS). To add further complexity,

Traveling Bax and Forth from Mitochondria to Control Apoptosis

Antiapoptotic Bcl-2 proteins on mitochondria inhibit prodeath proteins, such as Bax, which are found primarily in the cytosol. In this issue, Edlich et al., (2011) show that Bax and Bcl-xL interact on the mitochondrial surface and then retrotranslocate to the cytosol, effectively preventing Bax-induced permeabilization of mitochondria.

MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a ∼62u2009kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in ∼5 and ∼11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

Targeting Cell Death

Functional consequences of myocardial or cerebral infarction are the result of excessive cell death. It is patent that preventing cell death is the therapeutic goal in any ischemia‐reperfusion setting. Mitochondria amplify apoptotic cascades and have emerged as crucial organelles in ischemia‐reperfusion. Changes in mitochondrial inner membrane permeability and in the morphology of the organelle are regulated, perhaps interconnected processes that are starting to emerge as novel therapeutic targets for reducing cell death induced by ischemia‐reperfusion.