Ai miei genitori, alla mia famiglia e a tutte le persone a me care!!!

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Ai miei genitori, alla mia famiglia e a tutte le persone a me care!!! La vita è unita se si mette il cuore in quello che si fa, il cuore non come sentimento, ma come desiderio insopprimibile di felicità,
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Ai miei genitori, alla mia famiglia e a tutte le persone a me care!!! La vita è unita se si mette il cuore in quello che si fa, il cuore non come sentimento, ma come desiderio insopprimibile di felicità, di bene, di verità, di giustizia. Quel desiderio che hai sempre e a cui da solo non puoi dare piena risposta. Ci vuole Qualcosa di più grande per essere liberi. Bisogna che questo Qualcosa di più grande sia un esperienza, sia Qualcuno presente cui si risponde, sempre, in ogni momento della giornata. Tutto nella vita deve tendere a quel Qualcosa di più grande! (dai Discorsi di Enzo Piccinini) 1 2 ABSTRACT... 5 RIASSUNTO... 7 INTRODUCTION... 9 CA 2+ SIGNALING... 9 General overview... 9 Mitochondrial Ca 2+ signaling ER-mitochondria contact sites in Ca 2+ signaling The mitochondrial Ca 2+ uniporter (MCU) AUTOPHAGY Ca 2+ dependent control of autophagy APOPTOSIS MITOCHONDRIAL DISEASES Our experimental model: a MELAS patient with ND5 (13514A G) mutation CA 2+ SIGNALING IN NEURODEGENERATION AIMS RESULTS MITOCHONDRIAL DISEASES ND5 mutated fibroblasts present an increased autophagosome number already in basal conditions The increased autophagosome number in patient cells is not due to a block of the autophagic flux Mitochondria are direct substrates of autophagy in patient fibroblasts ND5 mutated fibroblasts show an alteration selectively in mitochondrial Ca 2+ homeostasis ND5 mutated fibroblasts are protected from apoptosis Patient cells do not present alterations in mitochondrial morphology and membrane potential, but show a clear deficiency in ER-mitochondria contact sites MCU overexpression induces a reduction in autophagosome number in patient fibroblasts The AMPK pathway is involved in the regulation of autophagic flux in mutated fibroblasts DISCUSSION MITOCHONDRIAL DISEASES RESULTS NEURODEGENERATION MCU overexpression enhances mitochondrial Ca 2+ uptake in primary cortical neurons MCU overexpression induces mitochondrial fragmentation MCU overexpression impairs neurons survival MCU-overexpression accelerates the loss of mitochondrial membrane potential in primary neurons MCU-overexpression elevates cytosolic Ca 2+ inducing excitotoxicity MCU-overexpression in vivo induces brain tissue degeneration DISCUSSION NEURODEGENERATION MATERIALS AND METHODS Cell culture, transfection and proteomic analysis Adenovirus production Aequorin Ca 2+ measurements FRET Ca 2+ measurements Mitochondrial membrane potential measurements ER-mitochondria colocalization Immunofluorescence Apoptotic counts Stereotaxic injection REFERENCES 4 ABSTRACT Ca 2+ is one of the main second messengers of cells and, in particular the Ca 2+ signaling in mitochondria is involved in different physiological processes spanning from cell metabolism, through the control of mitochondrial respiration and crucial metabolic enzymes, to the response in stress conditions. Despite the lack of a mechanistic understanding, it is well known that mitochondrial Ca 2+ overload is the most important trigger for the opening of permeability transition pore responsible for apoptosis induction after several toxic challenges. On the contrary, the role of Ca 2+ signaling in autophagy only recently started to emerge. Autophagy is a process of selfeating by which cellular organelles and proteins are sequestered and degraded in order to produce energy and amino acids in metabolic stress conditions, such as nutrient deprivation. It is not surprising that mitochondrial Ca 2+ also plays an important role in the pathological alteration of cell physiology in different human disorders. In the present work we will consider, in particular, the involvement of mitochondrial Ca 2+ homeostasis and its correlated metabolic processes in two models of human diseases: mitochondrial disorders and neurodegeneration. Mitochondrial disorders are a large group of heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation defects. We used skin primary fibroblasts derived from a patient with a complex I mutation in ND5 subunit, as a model of mitochondrial disorders. This system revealed an interesting correlation between the decrease in mitochondrial Ca 2+ uptake and the increase in autophagic flux. In addition, our results suggest that this is due to a structural rearrangement of intracellular organelle architecture causing a loss of ER-mitochondria contact sites. 5 Neurodegeneration is caused by selective and progressive death of specific neuronal subtypes. In order to understand the involvement of mitochondrial Ca 2+ signaling in the pathogenesis of neurodegeneration, we developed an in vitro system of mouse primary cortical neurons and we optimized an in vivo model of microinjection in mouse brain regions. In particular, we studied the effect of an increased mitochondrial Ca 2+ uptake, induced by the overexpression of mitochondrial Ca 2+ uniporter (MCU, the main responsible of Ca 2+ entry in mitochondrial matrix), on cell survival, in both primary cultures and in midbrain mouse area. We concluded that mitochondrial Ca 2+ accumulation induces mitochondrial fragmentation and higher sensitivity to cell death in neurons both in vitro and in vivo. 6 RIASSUNTO Il Ca 2+ è uno dei principali secondi messaggeri cellulari, ed in particolare il segnale Ca 2+ mitocondriale è implicato in vari processi fisiologici che spaziano dal metabolismo, attraverso il controllo della respirazione mitocondriale, alla risposta a condizioni di stress. Nonostante alcuni meccanismi d azione non siano ancora stati chiariti, il ruolo del Ca 2+ nell attivazione del processo apoptotico è ampiamente riconosciuto e comprovato. Al contrario, il coinvolgimento del segnale Ca 2+ in un altro importante processo, quale quello autofagico, ha cominciato ad emergere solo recentemente. Il ruolo del Ca 2+ a livello fisiologico risulta dunque fondamentale all interno della cellula e alterazioni nella sua regolazione hanno ripercussioni così profonde da indurre l'evolversi di differenti patologie umane. Nel presente lavoro verrà approfondito il ruolo del Ca 2+ mitocondriale in particolar modo in due modelli di patologie umane: le malattie mitocondriali e la neurodegenerazione. Le malattie mitocondriali sono un gruppo molto eterogeneo di patologie, accomunate principalmente dalla perdita di funzionalità della catena respiratoria. Come modello di studio di queste patologie abbiamo scelto di utilizzare delle colture primarie di fibroblasti umani derivanti da pazienti con una specifica mutazione nel gene per la subunità ND5 del complesso I della catena respiratoria del DNA mitocondriale. L utilizzo di questo modello sperimentale si è rivelato molto utile per l identificazione di una interessante correlazione tra la diminuzione dell uptake di Ca 2+ mitocondriale e l aumento del flusso autofagico in queste cellule. Inoltre, i nostri risultati suggeriscono che la causa del ridotto accumulo di Ca 2+ mitocondriale è direttamente correlato con un riarrangiamento spaziale nella distribuzione di reticolo endoplasmatico e mitocondri, tale per cui i siti di contatti presenti tra questi due organelli diminuiscono nettamente. 7 La neurodegenerazione è causata dalla selettiva e progressiva perdita di specifici tipi neuronali. Allo scopo di studiare il coinvolgimento del Ca 2+ nella neurodegenerazione, abbiamo sviluppato un modello in vitro di neuroni primari di corteccia di topo, in cui abbiamo analizzato gli effetti della sovraespressione del canale per il Ca 2+ mitocondriale, MCU (mitochondrial Ca 2+ uniporter). Dai nostri dati possiamo concludere che la sovraespressione di MCU ha degli effetti dannosi per le cellule neuronali, tanto da indurne la morte. Inoltre, abbiamo dei risultati preliminari anche in un sistema in vivo, i quali confermano e consolidano i dati ottenuti in vitro. Nello specifico, abbiamo iniettato vettori adeno-virali esprimenti il canale del Ca 2+ mitocondriale nel mesencefalo di topo, utilizzando la tecnica dell iniezione stereotassica, ed anche in questo caso osserviamo l induzione di morte cellulare e degenerazione neuronale. 8 INTRODUCTION Ca 2+ SIGNALING General overview Intracellular signaling requires second messengers, whose concentration rapidly and efficiently varies with time, it follows that one of the most important cellular messengers is Ca 2+. Indeed, between cytosol and extracellular environment there are both chemical and electrochemical gradients. Cells invest much of their ATP energy to affect changes in [Ca 2+ ] in space and time (Clapham, 2007). These rapid modifications in intracellular [Ca 2+ ] require the binding to buffering proteins, the compartmentalization into intracellular stores or the extrusion outside the cell (Berridge, 2009). Ca 2+ binding triggers changes in protein shape and charge and consequently activates or inhibits protein functions. The best known protein that buffers Ca 2+ is calmodulin. This buffering protein and others can control the amplitude and the timing of Ca 2+ signaling (Hoeflich and Ikura, 2002). Ca 2+ signaling in cells consists in dynamic variations of the cytosolic [Ca 2+ ], which at basal level is very low, even small fluctuations are sufficient to induce significant modifications. Cellular Ca 2+ fluxes relay on two main sources: the extracellular medium and the internal stores. The most important Ca 2+ store in the cell is the Endoplasmic Reticulum (ER), but recent works demonstrated that also other organelles, such as Golgi apparatus, endosome and lysosome are able to participate in Ca 2+ signaling (Pinton et al., 1998) (Calcraft et al., 2009). The signals that triggers Ca 2+ changes generate Ca 2+ waves within the cytoplasm, where it can stimulate numerous physiological Ca 2+ -sensitive processes, like muscle contraction, hormone secretion, synaptic transmission, cellular proliferation, apoptosis and others (Berridge et al., 2000) (Hajnoczky et al., 1995) (Rizzuto, 2003). 9 Cells use different types of mechanisms to access to the different Ca 2+ sourses. These pathways are not exclusive and most cells express combination of them. The best known pathway involves the release of IP3 after stimulation with a hormone, and the consequent release of Ca 2+ from the ER through the binding to the IP3R. Once Ca 2+ has carried out its signaling functions, it is rapidly extruded from the cytoplasm by various pumps and exchangers, and intracellular [Ca 2+ ] returns to resting conditions. The extrusion from the cells or the compartmentalization of Ca 2+ is due to the action of ATPase pumps, that use ATP like energy sources to maintain low intracellular [Ca 2+ ] by extruding Ca 2+ from the cells or into intracellular Ca 2+ stores. Given that the message decoded by Ca 2+ is given to the cells like an oscillatory difference of [Ca 2+ ], it is simple to understand the high complexity of pumps and channels that, with their activity, modulate the Ca 2+ message. During last decades, many scientists focused their attention on the identification of all the import/outport mechanisms for Ca 2+ signaling, but in spite of this large effort, the whole scenario is not yet completely clear. Mitochondrial Ca 2+ signaling Mitochondria had an important role in the evolution of the eukaryotic cells. These organelles are characterized by a particular structure. They are double membrane-bounded organelles thought to be derived from an proteobacterium-like ancestor, presumably due to a single ancient invasion occurred more than 1.5 billion years ago. The basic evidence of this endosymbiont theory (Dyall et al., 2004) is the existence of the mitochondrial DNA (mtdna), with structural and functional analogies to bacterial genomes. Mitochondria are defined by two structurally and functionally different membranes: outer membrane (OMM) and the inner membrane (IMM), characterized by invaginations called cristae, which enclose the mitochondrial matrix. The space between these two structures is traditionally called intermembrane space (IMS), but recent advances in electron microscopy techniques shed new light on the complex topology of the inner membrane. Cristae indeed are 10 not simply random folds, but rather internal compartments are formed by profound invaginations, originating from very tiny point-like structures in the inner membrane (Mannella, 2006). These narrow tubular structures, called cristae junctions, can limit the diffusion of molecule from the intra-cristae space towards the IMS, thus creating a microenvironment where respiratory chain complexes, and also other proteins, are hosted and protected from random diffusion. The OMM contains high copy number of a specific transport protein, VDAC (Voltage- Dependent Anion Channel), which is able to form pores on the membrane, becoming mostly permeable to ions and metabolites up to 5000 Da. However, the IMM is a highly selective membrane, thanks to the presence of cardiolipin, specific phospholipid that make the membrane permeable only to some ions. In addition, on the IMM it is possible to find also other specific transport proteins. The chemiosmotic theory of energy transfer was first demonstrated by Mitchell (Mitchell, 1967), who showed that the electrochemical gradient across the IMM is utilized by the F1/F0 ATPase to convert the energy of NADH and FADH 2, generated by the breaking down of energy rich molecules, such as glucose, into ATP. This gradient is characterized, for the most part, by electrical charge across the membrane ( ) and, in minor part it is a H + concentration difference between the two compartments ( ph). These differences of membrane potential generate a huge driving force that allows the passage of cations through the low sensitive Ca 2+ channels into the matrix. This gradient is normally maintained in the range of -120/-200 mv. Mitochondria are very important components of intracellular Ca 2+ signaling. Inside mitochondria Ca 2+ regulates firstly the production of ATP, by the mitochondrial respiratory chain, determining the rate of ATP production (McCormack et al., 1990); in addition, it triggers cellular metabolic adaptation to nutrient levels and it could initiate apoptosis after specific stimuli (Rasola and Bernardi, 2011). Different [Ca 2+ ] in the mitochondrial matrix regulates aerobic metabolism, tuning mitochondrial ATP production in 11 the needs of a stimulated cell by the control of metabolic enzymes. There are two Kreb cycle s dehydrogenases (isocitrate deidrogenase and -ketoglutarato deidrogenase) that are Ca 2+ -sensitive since they directly bind Ca 2+ and pyruvate dehydrogenase undergoing a dephosphorylating step in a Ca 2+ -dependent manner (Melendez-Hevia et al., 1996). Thus, the increase in Ca 2+ level into the matrix modulates the activity of Kreb cycle s enzymes and therefore the passage of electrons through the respiratory chain with the subsequent generation of the gradient across the IMM, which is necessary for ATP production. When Ca 2+ has carried out its functions in the mitochondria, it is necessary to rapidly extrude it in order to renew the resting balance into mitochondria. Ca 2+ extrusion is finely regulated by different exchangers, that are Na+/Ca 2+ or H+/Ca 2+ exchangers (Palty et al., 2010). If this mechanism for the regulation of mitochondrial [Ca 2+ ] fails and high levels of Ca 2+ are reached in the mitochondria, apoptosis is initiated. These conclusions started from the observation that Bcl-2 has a role in the modulation of Ca 2+ ions fluxes (Pinton and Rizzuto, 2006). This protein, like other anti-apoptotic proteins, reduces mitochondrial Ca 2+ response to extracellular stimuli by reducing the ER Ca 2+ levels. On the other hand, pro-apoptotic proteins exert their effect by increasing mitochondrial sensitivity. Massive Ca 2+ entry into mitochondria causes PTP opening that leads to modifications in mitochondrial morphology and the release of pro-apoptotic factors, such as cytochrome c, that initiate the complex cascade of apoptosis. ER-mitochondria contact sites in Ca 2+ signaling A key feature of mitochondria is their spatial organization in the cell. They are not solitary organelles, but they make contact with several other structures, among which the ER has obtained the most attention. Indeed, the physical and functional coupling of these two organelles in living cells, was originally found to determine the transfer of Ca 2+ between the two organelles. 12 There are several works in which was underlined the presence of overlapping regions of two organelles (thus establishing an upper limit of 100 nm for their distance) and allowed to estimate the area of the contact sites as 5-20% of total mitochondrial surface (Rizzuto et al., 1993; Rizzuto et al., 1992) (Rizzuto et al., 1998). More recently, electron tomography techniques allowed to estimate an even smaller distance (10-25 nm), as well as the presence of trypsin-sensitive tethers between the two membranes (Csordas et al., 2006). In mammals, many proteins have been identified to be indirectly involved in the regulation of ER mitochondria functional interaction, such as some chaperones, PACS-2, BAP31 and NOGO-A. In the search for the long-sought direct tether, Scorrano and coworkers have recently identified Mfn2 as the first mammalian protein to directly bridge the two organelles. It is retrieved from both ER and mitochondria, and it regulates their morphology. Mfn2 is rich in the ER mitochondria interface and connects ER with mitochondria via direct interactions between the protein localized in the ER and Mfn1 or Mfn2 present in the OMM. They also showed that genetic ablation of Mfn2 causes an increase in the distance between the two organelles with a consequent impairment of mitochondrial Ca 2+ uptake, thus further supporting the high [Ca 2+ ] microdomains theory (de Brito and Scorrano, 2008). The role of Mfn2 in tethering the two organelles was also confirmed in different systems (Wasilewski et al., 2012) (Area-Gomez et al., 2012). The mitochondrial Ca 2+ uniporter (MCU) During the past, the study of the cellular processes mediated by mitochondrial Ca 2+ was severely limited by the lack of the molecular identity of the channel responsible of Ca 2+ entry into the organelle. A lot of attempts have been made during the decades and several yet another mitochondrial Ca 2+ uniporter have been identified, but without success. Each of them presented critical points that lead these hypothesis to disappear from the scene. 13 The first important step was obtained from Clapham s group in the 2004, they for first hypothesized and demonstrated the channel s nature of the mitochondrial Ca 2+ uptake system, that they called MiCa (Kirichok et al., 2004). Nevertheless this important discovery about MCU s nature, the molecular identity of this channel remained unresolved. The only thinks known for years were the physical properties of the channel, its dependence on mitochondrial membrane potential, its sensitivity to Ruthenium Red and its activity when extramitochondrial [Ca 2+ ] is in the micromolar range. Subsequently, Graier s group proposed a role of mammalian uncoupling protein (UCP) in the mitochondrial Ca 2+ uniporter. However, the first results obtained from Trenker et al. have not been confirmed by other groups and also this hypothesis disappeared from the scene (Trenker et al., 2007). In the 2009, Clapham s group tried again to address the issue with a careful genome-wide Drosophila RNA interference (RNAi) screening. The human homolog of CG4589 Letm1 (leucine zipper EF-hand-containing transmembrane protein 1) is an highly conserved homomeric protein that is selectively localized on the inner mitochondrial membrane. In presence of low mitochondrial [Ca 2+ ], it imports Ca 2+ into mitochondria, representing an important component of the Ca 2+ entry machinery, but not like so much wanted MCU (Jiang et al., 2009). Another important study that has allowed to better understand the mitochondrial Ca 2+ uptake machinery was the
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