A Novel Ca2+ Release Channel in Mitochondria of Drosophila melanogaster: Properties and Role in Ca2+ Homeostasis

Mitochondrial Ca2+ uptake and release play a pivotal role in different physiological processes such as intracellular Ca2+ signaling and cell metabolism, while their dysregulation can lead to cell death induction. In energized mitochondria the Ca2+ uniporter (MCU) mediates Ca2+ uptake across the inner mitochondrial membrane (IMM) while the Na+/Ca2+ exchanger and the H+/Ca2+ exchanger are responsible for Ca2+ efflux. However, when mitochondrial matrix Ca2+ load exceeds the capacity of the efflux pathway by exchangers, an additional pathway for Ca2+ release from mitochondria may exist through opening of the permeability transition pore (PTP). The mitochondrial permeability transition (PT) describes a process of Ca2+-dependent, tightly regulated increase in the permeability of the IMM to solutes with molecular masses below 1500 Da, due to the opening of a high-conductance channel, the PTP. Prolonged PTP opening causes several effects, such as depolarization, osmotic swelling, outer mitochondrial membrane rupture and release of apoptogenic proteins like cytochrome c (cyt c). Transient openings of the PTP on the other hand, might be involved in physiological Ca2+ homeostasis and may protect mitochondria from Ca2+ overload. Several studies allowed a thorough characterization of the functional properties and regulation of the putative channel, but its molecular nature remains still elusive. One of the best defined modulators of the PTP is the mitochondrial peptidyl-prolyl-cis-trans-isomerase (PPIase) Cyclophilin D (Cyp-D), that plays an important role in protein folding and can be selectively inhibited by the immunosuppressant drug Cyclosporin A (CsA). In spite of its importance as a model organism and as a genetic tool, remarkably little is known about the properties of Ca2+ transport in mitochondria of the fruit fly Drosophila melanogaster, and on whether these mitochondria can undergo a PT. In this study we have characterized the pathways of Ca2+ transport in the digitonin-permeabilized embryonic Drosophila cell line S2R+. We demonstrated the presence of ruthenium red-sensitive Ca2+ uptake, and of Na+-stimulated Ca2+ release in energized mitochondria, which match well characterized Ca2+ transport pathways of mammalian mitochondria. Furthermore we identified and characterized a novel mitochondrial Ca2+-dependent Ca2+ release channel in Drosophila. Like the mammalian PTP, Drosophila Ca2+ release is inhibited by tetracaine and opens in response to matrix Ca2+ loading, IMM depolarization and thiol oxidation. As the yeast pore (and at variance from the mammalian PTP), the Drosophila channel is inhibited by Pi and insensitive to CsA. A striking difference between the pore of Drosophila and that of mammals is its selectivity to Ca2+ and H+ and the lack of mitochondrial swelling and cyt c release during the opening of the channel. The apparent absence of a mitochondrial Cyp in Drosophila prevents an investigation based on the effects of CsA, a classical inhibitor of the mammalian PTP. Thus, in the second part of this study we expressed human Cyp-D in Drosophila S2R+ cells in order to investigate its impact on the Ca2+-induced Ca2+ release channel. Preliminary Ca2+ retention capacity studies demonstrated that human Cyp-D induces opening of the Drosophila Ca2+ release channel in a rotenone-sensitive but CsA-insensitive manner. If the Cyp-D in Drosophila cells changes selectivity, size and properties of the Ca2+-induced Ca2+ release channel can now be addressed. We conclude that Drosophila mitochondria possess a selective Ca2+ release channel with features intermediate between yeast and mammals that is probably involved in Ca2+ homeostasis but not in Ca2+-mediated cell death induction. In this study we paved the way for the application from the sophisticated genetic strategies that Drosophila provides to define the molecular nature of the PTP and its role in pathophysiology of Ca2+ homeostasis

USP14 inhibition corrects an in vivo model of impaired mitophagy

Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin-proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well-characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14-induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age-related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. Our study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD-related symptoms

F-ATPase ofDrosophila melanogasterForms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+Release

Mitochondria of Drosophila melanogaster undergo Ca2+-induced Ca2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca2+ and H+. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg2+/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R+ cells sensitizes the mCrC to Ca2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca2+ and thiol oxidants and inhibited byMg(2+)/gamma-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species

Comprehensive Genetic Characterisation of Mitochondrial Ca2+ Uniporter Components Reveals Their Different Physiological Requirements in Vivo

Mitochondrial Ca2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca2+ uniporter has opened it up to genetic analysis in model organisms. Here, we report a comprehensive genetic characterisation of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmental lethal, consistent with unregulated Ca2+ uptake. Mutants for the neuronally-restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data interrogates the interplay between components of the mitochondrial Ca2+ uniporter, and sheds light on their physiological requirements in vivo.This work is supported by MRC core funding (MC_UU_00015/4, MC-A070-5PSB0 and MC_UU_00015/6) and ERC Starting grant (DYNAMITO; 309742) to A.J.W., and the Italian Ministry of Health “Ricerca Finalizzata” [GR-2011-02351151] to E.Z. T.P.G. and J.J.L. are supported by MRC Studentships awarded via the MRC MBU. V.L.H. was funded by an EMBO Long-Term Fellowship (ALTF 740-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409). Stocks were obtained from the Bloomington Drosophila Stock Center which is supported by grant NIH P40OD018537, and material was obtained from the Drosophila Genomics Resource Center, which is supported by NIH grant 2P40OD010949

Comprehensive Genetic Characterization of Mitochondrial Ca2+ Uniporter Components Reveals Their Different Physiological Requirements In Vivo.

Mitochondrial Ca2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca2+ uniporter has opened it up to genetic analysis in model organisms. Here, we report a comprehensive genetic characterization of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca2+ uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data reveal the interplay among components of the mitochondrial Ca2+ uniporter and shed light on their physiological requirements in vivo.This work is supported by MRC core funding (MC_UU_00015/4, MC-A070-5PSB0, and MC_UU_00015/6) and an ERC starting grant (DYNAMITO; 309742) to A.J.W., as well as by the Italian Ministry of Health “Ricerca Finalizzata” (GR-2011-02351151) to E.Z. T.P.G. and J.J.L. are supported by MRC studentships awarded via the MRC MBU. V.L.H. was funded by an EMBO Long-Term Fellowship (ALTF 740-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409)

Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction.

Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.his work was supported by grants from the Italian Ministry of Health “Ricerca Finalizzata” (GR-2011-02351151), Rita Levi Montalcini “Brain Gain” program, and Michael J Fox RRIA 2014 (Grant ID 9795) to E Ziviani and by ERC FP7-282280, FP7 CIG PCIG13-GA-2013-618697, and Italian Ministry of Research FIRB RBAP11Z3YA_005 to L Scorrano. AJ Whitworth is funded by MRC Core funding (MC_UU_00015/6)

A Novel Ca2+ Release Channel in Mitochondria of Drosophila melanogaster: Properties and Role in Ca2+ Homeostasis

Mitochondrial Ca2+ uptake and release play a pivotal role in different physiological processes such as intracellular Ca2+ signaling and cell metabolism, while their dysregulation can lead to cell death induction. In energized mitochondria the Ca2+ uniporter (MCU) mediates Ca2+ uptake across the inner mitochondrial membrane (IMM) while the Na+/Ca2+ exchanger and the H+/Ca2+ exchanger are responsible for Ca2+ efflux. However, when mitochondrial matrix Ca2+ load exceeds the capacity of the efflux pathway by exchangers, an additional pathway for Ca2+ release from mitochondria may exist through opening of the permeability transition pore (PTP). The mitochondrial permeability transition (PT) describes a process of Ca2+-dependent, tightly regulated increase in the permeability of the IMM to solutes with molecular masses below 1500 Da, due to the opening of a high-conductance channel, the PTP. Prolonged PTP opening causes several effects, such as depolarization, osmotic swelling, outer mitochondrial membrane rupture and release of apoptogenic proteins like cytochrome c (cyt c). Transient openings of the PTP on the other hand, might be involved in physiological Ca2+ homeostasis and may protect mitochondria from Ca2+ overload. Several studies allowed a thorough characterization of the functional properties and regulation of the putative channel, but its molecular nature remains still elusive. One of the best defined modulators of the PTP is the mitochondrial peptidyl-prolyl-cis-trans-isomerase (PPIase) Cyclophilin D (Cyp-D), that plays an important role in protein folding and can be selectively inhibited by the immunosuppressant drug Cyclosporin A (CsA). In spite of its importance as a model organism and as a genetic tool, remarkably little is known about the properties of Ca2+ transport in mitochondria of the fruit fly Drosophila melanogaster, and on whether these mitochondria can undergo a PT. In this study we have characterized the pathways of Ca2+ transport in the digitonin-permeabilized embryonic Drosophila cell line S2R+. We demonstrated the presence of ruthenium red-sensitive Ca2+ uptake, and of Na+-stimulated Ca2+ release in energized mitochondria, which match well characterized Ca2+ transport pathways of mammalian mitochondria. Furthermore we identified and characterized a novel mitochondrial Ca2+-dependent Ca2+ release channel in Drosophila. Like the mammalian PTP, Drosophila Ca2+ release is inhibited by tetracaine and opens in response to matrix Ca2+ loading, IMM depolarization and thiol oxidation. As the yeast pore (and at variance from the mammalian PTP), the Drosophila channel is inhibited by Pi and insensitive to CsA. A striking difference between the pore of Drosophila and that of mammals is its selectivity to Ca2+ and H+ and the lack of mitochondrial swelling and cyt c release during the opening of the channel. The apparent absence of a mitochondrial Cyp in Drosophila prevents an investigation based on the effects of CsA, a classical inhibitor of the mammalian PTP. Thus, in the second part of this study we expressed human Cyp-D in Drosophila S2R+ cells in order to investigate its impact on the Ca2+-induced Ca2+ release channel. Preliminary Ca2+ retention capacity studies demonstrated that human Cyp-D induces opening of the Drosophila Ca2+ release channel in a rotenone-sensitive but CsA-insensitive manner. If the Cyp-D in Drosophila cells changes selectivity, size and properties of the Ca2+-induced Ca2+ release channel can now be addressed. We conclude that Drosophila mitochondria possess a selective Ca2+ release channel with features intermediate between yeast and mammals that is probably involved in Ca2+ homeostasis but not in Ca2+-mediated cell death induction. In this study we paved the way for the application from the sophisticated genetic strategies that Drosophila provides to define the molecular nature of the PTP and its role in pathophysiology of Ca2+ homeostasis.L’accumulo e il rilascio di Ca2+ da parte dei mitocondri svolge un ruolo centrale in diversi processi fisiologici, come nelle vie di segnalazione intracellulari e nel metabolismo cellulare, mentre la loro disregolazione può indurre la morte cellulare. In mitocondri energizzati, l’uniporto del Ca2+ (MCU) media l’accumulo di Ca2+ attraverso la membrane mitocondriale interna (MMI) mentre gli scambiatori Na+/Ca2+ e H+/Ca2+ sono responsabili del suo efflusso. Tuttavia, quando il carico di Ca2+ nella matrice mitocondriale supera la capacità di efflusso attraverso gli scambiatori, potrebbe attivarsi una via aggiuntiva di rilascio di Ca2+, attraverso l’apertura del poro di transizione di permeabilità (PTP). La transizione di permeabilità (TP) consiste nell’aumento della permeabilità della MMI a soluti con massa molecolare inferiore a 1500 Da, è un processo Ca2+-dipendente, strettamente regolato, e dovuto all’apertura di un canale ad alta conduttanza, il PTP. La prolungata apertura del PTP provoca diversi effetti, come la depolarizzazione, il rigonfiamento osmotico, la rottura della membrana mitocondriale esterna e il rilascio di proteine pro-apoptotiche come il citocromo c (cit c). L’apertura transiente del PTP, invece potrebbe essere coinvolta nell’omeostasi fisiologica del Ca2+ e potrebbe proteggere i mitocondri da un sovraccarico dello stesso. Diversi studi hanno consentito una caratterizzazione accurata delle proprietà funzionali e della regolazione del canale putativo, ma la sua natura molecolare rimane ignota. Uno dei miglior modulatori caratterizzati del PTP è la peptidil-prolil-cis-trans-isomerasi mitocondriale Ciclofilina D (Cip-D), che svolge un ruolo importante nel ripiegamento delle proteine e può essere selettivamente inibita dal farmaco immunosoppressore Ciclosporina A (CsA). Nonostante la sua importanza come organismo modello e come strumento genetico, poco si conosce a proposito delle proprietà di trasporto di Ca2+ mitocondriale del moscerino della frutta Drosophila melanogaster e dell’eventualità che i suoi mitocondri possano subire una TP. In questo studio abbiamo caratterizzato le vie di trasporto di Ca2+ mitocondriale nella linea cellulare embrionica di Drosophila S2R+, permeabilizzata con la digitonina. Abbiamo dimostrato la presenza di un effettivo accumolo di Ca2+, sensibile al rosso rutenio, come anche il rilascio di Ca2+ stimolato dal Na+ in mitocondri energizzati, processi che corrispondono alle ben caratterizzate vie di trasporto di Ca2+ in mammiferi. Inoltre, abbiamo identificato e caratterizzato un nuovo canale di rilascio del Ca2+ indotto dal Ca2+ stesso in Drosophila. Come il PTP dei mammiferi, il canale di rilascio di Ca2+ in Drosophila è inibito da tetracaina e apre in risposta al carico di Ca2+ nella matrice, depolarizzazione della MMI ed ossidazione di tioli. Come il poro in lievito (e in contrasto con il PTP dei mammiferi), il poro di Drosophila è inibito da Pi e insensibile alla CsA. Le principali differenze il poro di Drosophila e quello dei mammiferi sono la sua selettività per Ca2+ e H+, e la mancanza di rigonfiamento mitocondriale e il rilascio di cit c conseguente all’apertura del canale. L’apparente assenza di una Cip mitocondriale in Drosophila impedisce uno studio basato sugli effetti della CsA, un classico inibitore del PTP dei mammiferi. Perciò, nella seconda parte di questo studio abbiamo espresso la Cip-D umana nelle cellule S2R+ di Drosophila al fine di indagare il suo impatto sul canale di rilascio del Ca2+. Preliminari studi di capacità di ritenzione del Ca2+ hanno dimostrato che la Cip-D umana induce l’apertura del canale di rilascio del Ca2+ in Drosophila in modo rotenone-sensibile ma CsA-insensibile. Se la Cip-D in Drosophila cambia la selettività, la grandezza e le proprietà del canale di rilascio del Ca2+ può ora essere investigato. Concludiamo che i mitocondri di Drosophila possiedono un canale selettivo di rilascio di Ca2+ con caratteristiche intermedie tra il lievito e i mammiferi che probabilmente è coinvolto nell’omeostasi del Ca2+, ma non nell’induzione della morte cellulare mediata dal Ca2+. In questo studio abbiamo spianato la strada per l’applicazione delle strategie di genetica sofisticate, che la Drosophila offre per definire la natura molecolare del PTP ed il suo ruolo nella patofisiologia dell’omeostasi del Ca2+

The permeability transition pore as a Ca2+ release channel: New answers to an old question

Mitochondria possess a sophisticated array of Ca2+ transport systems reflecting their key role in physiological Ca2+ homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca2+ uptake, the ruthenium red (RR)-sensitive mitochondrial Ca2+ uniporter (MCU); and one main mechanism for Ca2+ release, the RR-insensitive 3Na+-Ca2+ antiporter. An additional mechanism for Ca2+ release is provided by a Na+ and RR-insensitive release mechanism, the putative 3H+-Ca2+ antiporter. A potential kinetic imbalance is present, however, because the Vmax of the MCU is of the order of 1,400 nmol Ca2+ x mg-1 protein x min-1 while the combined Vmax of the efflux pathways is about 20 nmol Ca2+ x mg-1 protein x min-1. This arrangement exposes mitochondria to the hazards of Ca2+ overload when the rate of Ca2+ uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca2+]. In this short review we discuss the hypothesis that transient opening of the Ca2+-dependent permeability transition pore may provide mitocondria with a fast Ca2+ release channel preventing Ca2+ overload. We also address the relevance of a mitochondrial Ca2+ release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals