The multifunctional mitochondrial inner membrane protein Optic Atrophy 1 controls cellular damage in vivo

Mitochondria are crucial organelles for cell life and death. They represent the site of oxidative phosphorylation which provides energy for cellular processes, and play key roles in Ca2+ signaling and integration and amplification of apoptotic signals. Mitochondria are double membrane organelles: they possess an outer mitochondrial membrane (OMM), and an inner mitochondrial membrane (IMM) that can be further subdivided in an inner ’’boundary membrane’’ and in the cristae which are compartments separated from the inner boundary membrane by narrow tubular junctions. In addition mitochondria are highly dynamic and heterogeneous. Mitochondrial morphology and distribution are actively controlled to respond to the functional needs of the cell. This functional versatility is controlled by a growing family of ,,mitochondria-shaping’’ proteins that regulate fusion and fission events. In mammals mitochondrial fission depends on the cytoplasmic GTPase, dynamin related 1 (DRP1), and its adaptors on the outer mitochondrial membrane (OMM). Mitochondrial fusion is regulated by two OM proteins mitofusin 1and 2 (MFN1and MFN2), and Optic Atrophy 1 (OPA1) in the inner membrane (IM). OPA1 promotes mitochondrial fusion depending on MFN1 and has an antiapoptotic function genetically distinguishable from its role in fission. OPA1 controls apoptotic cristae remodeling, a process required for the complete release of cytochrome c, the only soluble component of the respiratory chain. OPA1 oligomerizes in high molecular weight complexes, required for the maintenance of the cristae junction tight, thus preventing cytochrome c mobilization. In vitro, disruption of these complexes by BH3 only proteins correlates with the widening of the narrow tubular junction and with the fusion of the individual cristae. Recently we identified a new role for OPA1 in the control of stability and assembly of the respiratory chain supercomplexes, mitochondrial respiratory efficiency and respiratory growth. However, very little is known about the role of OPA1 and of OPA1-dependent cristae changes in vivo. The aim of this thesis was to explore the role of OPA1 in cell death in vivo, by characterizing an OPA1 overexpressing (Opa1tg) mice generated in our laboratory where the Opa1 transgene is targeted to a specific position in the X chromosome and under the control of the weak ubiquitous human beta-actin promoter. By backcrossing mice in the genetic backgrounds C57/ Bl6 and SV129 we obtained inbred lines where OPA1 was mildly overexpressed in a variety of tissues. This slight increase in OPA1 levels did not alter other proteins involved in mitochondrial dynamics, and did not affect the development, life and fertility of the animals. We found that the life span of Opa1tg C57/Bl6 was not different from that of wild type (Wt) littermates, while it was significantly decreased in Opa1tg SV129, mostly due to the development of hematologic and solid tumors. Since Opa1tg mice did not show any overt phenotype, we decided to analyze morphologically and histological every organ. Hearts were bigger in Opa1tg animals 9 months after birth. Histological and immunofluorescence analysis did not reveal signs of hypertrophy and heart functionality was indeed preserved as demonstrated by ecocardiographyc analysis, indicating that the retrieved cardiac hypertrophy was not pathological and suggesting that perhaps aging-associated heart atrophy is inhibited in Opa1tg mice. To test if Opa1 overexpression interfered with the program of striated muscle atrophy, we turned to a well-established model of skeletal muscle atrophy, induced by sciatic nerve resection. Muscle atrophy was significantly reduced in Opa1tg mice, where mitochondrial function was preserved in the denervated fibers. This result suggested that Opa1 can interfere with at least one form of cell damage in vivo and prompted us to verify if it could interfere with other well established models of necrotic and apoptotic cell death. In a model of isolated perfused heart ischemia reperfusion, Opa1tg hearts were less susceptible to ischemic damage, and the size of the infarcted area was significantly reduces in Opa1tg mice following brain ischemic damage induced by Middle Cerebral Occlusion (MCAo). Finally, Fas induced hepatocellular apoptosis in vivo was drastically reduced in Opa1tg mice where hepatic damage was accordingly blunted. Mechanistically, muscle and liver mitochondria from Opa1tg mice were resistant to cristae remodeling and cytochrome c release induced by the pro-apoptotic BH3-only protein cBID and displayed increased efficiency of complex I dependent respiration, while other parameters of mitochondrial involvement in cell death were not affected. This increase in complex I dependent respiration correlated with an increase in Respiratory Chain Supercomplexes (RCS) that we demonstrated to be a consequence of OPA1-dependent cristae stabilization. Our data elucidate a role for cristae shape in different modes of cell death in vivo and point out a crucial role for mitochondrial ultrastructural remodeling in response to widespread pathological conditions, ranging from ischemia reperfusion to muscular atrophy. We can conclude that Opa1, cristae biogenesis and remodeling are central players of reversible and irreversible cell damage and death in vivo.

L’obbiettivo della tesi è stato rivolto all’ analisi del ruolo di OPA1 nella morte cellulare in vivo attraverso la caratterizzazione di un modello murino over-esprimente OPA1 (Opa1tg) generato nel nostro laboratorio; in tale modello il transgene Opa1 è stato inserito in una specifica posizione del cromosoma X e sotto il controllo del promotore debole ed ubiquitario per la beta-actina umana. Attraverso l’incrocio dei topi in due diversi background genetici C57/BL6 e SV129, abbiamo ottenuto linee murine in cui OPA1 è debolmente espressa in diversi tessuti. Il lieve incremento dei livelli di OPA1 non altera le altre proteine coinvolte nella dinamica mitocondriale e non ha alcun effetto sullo sviluppo, la longevità e la fertilità degli animali. Abbiamo osservato che la longevità dei topi Opa1tg C57/Bl6 non mostrava variazioni rispetto ai wild type (WT), mentre era notevolmente ridotta negli Opa1tg SV129, prevalentemente a causa di insorgenza di tumori ematologici e solidi. Poiché i topi Opa1tg non mostravano alcun evidente fenotipo, abbiamo deciso di effettuare un analisi morfologica ed istologica in ogni organo. I cuori degli animali Opa1tg all’età di 9 mesi erano più grandi. Le analisi istologiche e di immunofluorescenza non rivelavano segni di ipertrofia e la funzionalità cardiaca era invece preservata come dimostrato dalle analisi ecocardiografiche, indicando che l’ipertrofia cardiaca osservata non era patologica e suggerendo altresì che l’atrofia cardiaca associata all’invecchiamento era inibita nei topi Opa1tg. Per verificare se l’over-espressione di Opa1 interferiva in qualche modo con l’atrofia associata al muscolo striato, abbiamo utilizzato un noto modello di atrofia muscolare indotta tramite recisione del nervo sciatico. L’atrofia muscolare era significativamente ridotta nei topi Opa1tg, dove la funzionalità mitocondriale nelle fibre denervate era preservata. Questi risultati suggerivano che OPA1 può interferire con almeno una forma di danno cellulare in vivo e ci ha spinto a verificare se ciò potesse interferire anche in altri modelli conosciuti di morte cellulare quali necrosi ed apoptosi. In un modello di ischemia riperfusione di cuore isolato, i cuori Opa1tg erano meno suscettibili al danno ischemico e l’area d’infarto era significativamente ridotta nei topi Opa1tg dopo il danno da ischemia cerebrale indotto tramite l’Occlusione dell’arteria Cerebrale Mediale (MCAo). In fine, l’apoptosi epatocellulare in vivo indotta da Fas era drasticamente ridotta in topi Opa1tg , dove il danno epatico era diminuito. Meccanisticamente, i mitocondri da muscolo e fagato dei Opa1tg topi sono risultati resistenti al rimodellamento delle cristae ed al rilascio di citocromo c indotto dalla proteina proapoptotica cBID; mostrano inoltre una aumentata efficienza nella respirazione mediata dal complesso I, mentre glia altri parametri di morte cellulare mediata dai mitocondri non sono risultati alterati. L’aumento della respirazione dipendente da complesso I correla con un aumento dei Supercomplessi della Catena Respiratoria (RCS) che, come da noi dimostrato, è una conseguenza della stabilizzazione delle cristae dipendente da OPA1. I nostri dati dimostrano un ruolo della struttura delle cristae in diversi modelli di morte cellulare in vivo e comprovano un ruolo cruciale del rimodellamento dell’ultrastruttura mitocondriale in risposta a diverse condizioni patologiche, che vanno dall’ischemia-riperfusione all’atrofia muscolare. Possiamo concludere che OPA1, la biogenesi ed il rimodellamento delle cristae sono processi fondamentali di danno reversibile ed irreversibile e di morte cellulare in vivo.