Ruolo delle proteine alfa-sinucleina, parkina, DJ-1 e PINK1, mutate nelle forme familiari di Morbo di Parkinson, nel controllo dell'omeostasi mitocondriale dello ione Calcio

Neurodegenerative disorders, such as Parkinson’s diseases (PD), are characterized by loss of specific neuronal populations and have been associated with mitochondrial dysfunction and oxidative stress. In addition, endoplasmic reticulum (ER) stress, a severe alteration in the structure and function of the ER with the accumulation of misfolded proteins and alterations in calcium homeostasis, has been suggested to be involved in some neuronal diseases. Finally, disturbed functions of the ubiquitin proteasome system (UPS), responsible for the degradation of cytosolic, ER, and synaptic proteins, is known to contribute to ER stress. The mechanisms through which these alterations impact on cell function are complex. Among them, however, the effect on the spatio-temporal patterns of a key second messenger, such as Ca2+, most likely plays a key role. My PhD program aimed at clarifying this important issue, focusing on simple pathogenetic models. Indeed, although most forms of PD are sporadic and multifactorial, an increasing number of mutations in specific genes were found in rare, genetic forms of the disease. Understanding the molecular pathogenesis of these forms may give important clues to the understanding of the more common sporadic cases, as well as provide some novel insight into the basic cell biology mechanisms. Specifically, mutations in a set of genes (alpha-synuclein, parkin and most recently in DJ-1, PINK1, ubiquitin carboxyl-terminal hydrolase-1, LRRK2/dardarin and Omi/HTRA2) have been associated with familial cases of PD. In all cases, the normal function of the gene products is not fully understood. Our aim was to study the role of these gene products in subcellular Ca2+ homeostasis, with major focus on mitochondria. Mitochondria play a central role in cell biology not only as producers of ATP, but also in the sequestration of Ca2+. Since they are the major site of free radical production in cells, they are also a primary target for oxidative damage and subsequent dysfunction. Mitochondria are also repositories of several proteins which regulates apoptosis. Perturbations in the normal functions of mitochondria will inevitably disturb cell function, may sensitise cells to neurotoxic insults and may initiate cell death. Alterations in mitochondrial Ca2+ signalling could synergize with mitochondrial dysfunction in causing deleterious functional alterations and committing the cell to death. In addition to the analysis of Ca2+ homeostasis, we have analysed other aspects of mitochondrial physiology including the energetic metabolism and the relationship between mitochondria and ER. Common interesting aspects are emerging from the data presented in this thesis, which have considered both the overexpression and the silencing of alpha-synuclein, parkin, DJ-1 and PINK1 proteins: all of them are able to modulate mitochondrial Ca2+ homeostasis. In particular alpha-synuclein, parkin, DJ-1 operate through the same mechanism by enhancing the ER-mitochondria contact sites of about 10%, thus enhancing the Ca2+ transfer between the two organelles. When this action is missing, as we have documented in the case of alpha-synuclein, the autophagic process can be activated. As for PINK1 we have identified an interesting biphasic effect. We have now to clarify whether this action could be related to distinct PINK1 distribution on mitochondrial membranes in respect to its expression levels and whether it may act by regulating the activity of the mitochondrial Ca2+ toolkit proteins. Experiments performed in permeabilized cells exposed to fixed 1 microM Ca2+ concentration account for this possibility.Le malattie neurodegenerative, tra le quali il morbo di Parkinson (PD) sono associate a disfunzioni mitocondriali e a stress ossidativo. Inoltre, è stato suggerito che profonde alterazioni della struttura e delle funzioni del reticolo endoplasmatico (RE), una condizione nota come “ER stress”, e disfunzioni del sistema proteosoma-ubiquitina (UPS) siano alcuni dei processi cellulari coinvolti nell’insorgenza di queste malattie. I meccanismi attraverso i quali queste disfunzioni alterano la funzione cellulare sono complessi e solo parzialmente chiariti. Tra questi, gli effetti sulla distribuzione spazio-temporale di un secondo messaggero fondamentale per la corretta comunicazione cellulare, quale lo ione Ca2+, è sicuramente importante. Il programma del mio Dottorato di Ricerca prevedeva di chiarire alcuni di questi aspetti utilizzando modelli cellulari semplici. Infatti, anche se la maggior parte delle forme di PD sono sporadiche e multifattoriali, un numero crescente di mutazioni in geni specifici sono state individuate in alcune forme di malattia familiare. Lo studio delle funzioni delle proteine codificate da questi geni rappresenta quindi uno strumento molto importante per la comprensione degli aspetti molecolari alla base del processo neurodegenerativo. L’aspetto interessante è rappresentato dal fatto che le alterazioni cellulari alla base dei difetti genetici sono le stesse identificate nei casi sporadici. La delucidazione dei meccanismi patogenici delle forme genetiche, oltre a contribuire alla comprensione delle forme più diffuse, potrebbe quindi fornire anche nuove informazioni sui meccanismi cellulari alla base della insorgenza dei processi neurodegenerativi. In particolare, mutazioni in geni diversi (che codificano le proteine alfa-sinucleina, parkina, DJ-1, PINK1, ubiquitin carboxyl-terminal hydrolase-1 (UCHL-1), LRRK2/dardarina e Omi/HTRA2) sono state individuate in pazienti affetti da forme familiari di PD. In tutti i casi l’esatta funzione dei prodotti genici non è ancora completamente chiarita. Il programma di ricerca prevedeva di studiare il loro ruolo nella regolazione dell’omeostasi del Ca2+, con particolare attenzione alla funzione mitocondriale. Uno degli elementi comuni di molte malattie neurodegenerative è infatti rappresentato da disfunzioni del metabolismo mitocondriale del Ca2+. I mitocondri hanno un ruolo centrale nella biologia cellulare, non solo in quanto sono la sede principale di produzione di ATP, ma anche perché svolgono un ruolo importante nel sequestro del Ca2+ intracellulare e nella produzione di specie reattive dell’ossigeno e di radicali liberi. Essi sono il primo bersaglio del danno ossidativo e delle disfunzioni che ne derivano, inoltre contengono numerose proteine che regolano il fenomeno della morte cellulare per apoptosi. Perturbazioni della funzionalità mitocondriale portano quindi inevitabilmente a disturbi del funzionamento cellulare e possono dare origine al processo di morte cellulare rendendo le cellule più suscettibili agli insulti neurotossici. Oltre all’omeostasi mitocondriale del Ca2+ sono stati analizzati anche altri aspetti legati alla fisiologia mitocondriale, in particolare il metabolismo energetico e i rapporti tra mitocondri e RE. Dagli esperimenti presentati in questa tesi, che hanno analizzato sia gli effetti della sovraespressione che del silenziamento delle proteine alfa-sinucleina, parkina, DJ-1 e PINK1, emergono alcuni aspetti comuni molto interessanti: tutte queste proteine sono in grado di modulare l’omeostasi del Ca2+ mitocondriale. Per quanto riguarda alfa-sinucleina, parkina, DJ-1 abbiamo osservato che esse condividono anche il meccanismo con il quale operano questa modulazione: in tutti e tre i casi la loro sovraespressione provoca un aumento di circa il 10% dei contatti tra reticolo endoplasmatico e mitocondri e quindi potenzia il trasferimento di Ca2+ tra questi due organelli. Se questa funzione viene a mancare, come abbiamo dimostrato nel caso di alfa-sinucleina, viene attivata la risposta autofagica. Per quanto riguarda PINK1 abbiamo evidenziato la possibilità che possa regolare l’omeostasi mitocondriale del Ca2+ attraverso un’azione bifasica, resta da chiarire se ciò dipenda dalla sua distribuzione nelle membrane mitocondriali e dalla sua attività sulle proteine che regolano il trasporto del Ca2+ mitocondriale. Gli esperimenti condotti in cellule permeabilizzate, ed esposte ad una concentrazione di Ca2+ pari a 1 microM, sembrano suggerire questa possibilità

Mitochondrial Ca(2+) and neurodegeneration.

Mitochondria are essential for ensuring numerous fundamental physiological processes such as cellular energy, redox balance, modulation of Ca(2+) signaling and important biosynthetic pathways. They also govern the cell fate by participating in the apoptosis pathway. The mitochondrial shape, volume, number and distribution within the cells are strictly controlled. The regulation of these parameters has an impact on mitochondrial function, especially in the central nervous system, where trafficking of mitochondria is critical to their strategic intracellular distribution, presumably according to local energy demands. Thus, the maintenance of a healthy mitochondrial population is essential to avoid the impairment of the processes they regulate: for this purpose, cells have developed mechanisms involving a complex system of quality control to remove damaged mitochondria, or to renew them. Defects of these processes impair mitochondrial function and lead to disordered cell function, i.e., to a disease condition. Given the standard role of mitochondria in all cells, it might be expected that their dysfunction would give rise to similar defects in all tissues. However, damaged mitochondrial function has pleiotropic effects in multicellular organisms, resulting in diverse pathological conditions, ranging from cardiac and brain ischemia, to skeletal muscle myopathies to neurodegenerative diseases. In this review, we will focus on the relationship between mitochondrial (and cellular) derangements and Ca(2+) dysregulation in neurodegenerative diseases, emphasizing the evidence obtained in genetic models. Common patterns, that recognize the derangement of Ca(2+) and energy control as a causative factor, have been identified: advances in the understanding of the molecular regulation of Ca(2+) homeostasis, and on the ways in which it could become perturbed in neurological disorders, may lead to the development of therapeutic strategies that modulate neuronal Ca(2+) signaling

Measurements of Ca(2+) concentration with recombinant targeted luminescent probes.

In the last two decades the study of Ca(2+) homeostasis in living cells has been enhanced by the explosive development of genetically encoded Ca(2+)-indicators. The cloning of the Ca(2+)-sensitive photoprotein aequorin and of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been enormously advantageous. As polypeptides, aequorin and GFP allow their endogenous production in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localize them within the cell by including defined targeting signals in the amino acid sequence. These two proteins have been extensively engineered to obtain several recombinant probes for different biological parameters, among which Ca(2+) concentration reporters are probably the most relevant. The GFP-based Ca(2+) probes and aequorin are widely employed in the study of intracellular Ca(2+) homeostasis. The new generation of bioluminescent probes that couple the Ca(2+) sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca(2+) changes in single cell imaging experiments and the video-imaging of Ca(2+) concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes

Mitochondrial calcium homeostasis and implications for human health.

Ca2+ has a central role in all the cellular functions. Its signal is shaped by the coordinated action of the Ca2+ transporting proteins and the intracellular organelles. Mitochondria have a special role since they are the energy powerhouse of the cells, but also a major hub for cellular Ca2+ signaling crucial for cell life and death. The mitochondrial membrane potential generated by the respiratory chain is used by the ATP synthase for running the endergonic reaction of ADP phosphorylation and by the mitochondrial Ca2+ uniporter to take up Ca2+ into the matrix accordingly its electrochemical gradient. The action of the H+/Ca2+ and the Na+/Ca2+ exchangers prevents the attainment of the electrical equilibrium. Impaired Ca2+ handling can lead to matrix Ca2+ overload and activation of the high conductance mitochondrial permeability transition pore. Mitochondrial Ca2+ overload has deleterious consequences for the cells: increased membrane permeability leads to the release of pro-apoptotic factors and the activation of the apoptotic pathway. Even the absence of proper Ca2+ transfer from the endoplasmic reticulum to mitochondria could be detrimental since it results in defective metabolism and autophagy. Thus, mitochondrial Ca2+ handling dysfunctions may have important implications in different physio-pathological conditions

Mitochondrial Ca(2+) as a key regulator of mitochondrial activities.

Mitochondria play a central role in cell biology, not only as producers of ATP but also as regulators of the Ca(2+) signal. The translocation by respiratory chain protein complexes of H(+) across the ion-impermeable inner membrane generates a very large H(+) electrochemical gradient that can be employed not only by the H(+) ATPase to run the endoergonic reaction of ADP phosphorylation, but also to accumulate cations into the matrix. Mitochondria can rapidly take up Ca(2+) through an electrogenic pathway, the uniporter, that acts to equilibrate Ca(2+) with its electrochemical gradient, and thus accumulates the cation into the matrix, and they can release it through two exchangers (with H(+) and Na(+), mostly expressed in non-excitable and excitable cells, respectively), that utilize the electrochemical gradient of the monovalent cations to prevent the attainment of electrical equilibrium.The uniporter, due to its low Ca(2+) affinity, demands high local Ca(2+) concentrations to work. In different cell systems these high Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of the plasma membrane and the sarco/endoplasmic reticulum Ca(2+) channels.Recent work has revealed the central role of mitochondria in signal transduction pathways: evidence is accumulating that, by taking up Ca(2+), they not only modulate mitochondrial activities but also tune the cytosolic Ca(2+) signals and their related functions. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarize cell physiology aspects of the mitochondrial Ca(2+) transport machinery

Calcium signaling in Parkinson's disease.

Calcium (Ca2+) is an almost universal second messenger that regulates important activities of all eukaryotic cells. It is of critical importance to neurons, which have developed extensive and intricate pathways to couple the Ca2+ signal to their biochemical machinery. In particular, Ca2+ participates in the transmission of the depolarizing signal and contributes to synaptic activity. During aging and in neurodegenerative disease processes, the ability of neurons to maintain an adequate energy level can be compromised, thus impacting on Ca2+ homeostasis. In Parkinson's disease (PD), many signs of neurodegeneration result from compromised mitochondrial function attributable to specific effects of toxins on the mitochondrial respiratory chain and/or to genetic mutations. Despite these effects being present in almost all cell types, a distinguishing feature of PD is the extreme selectivity of cell loss, which is restricted to the dopaminergic neurons in the ventral portion of the substantia nigra pars compacta. Many hypotheses have been proposed to explain such selectivity, but only recently it has been convincingly shown that the innate autonomous activity of these neurons, which is sustained by their specific Cav1.3 L-type channel pore-forming subunit, is responsible for the generation of basal metabolic stress that, under physiological conditions, is compensated by mitochondrial buffering. However, when mitochondria function becomes even partially compromised (because of aging, exposure to environmental factors or genetic mutations), the metabolic stress overwhelms the protective mechanisms, and the process of neurodegeneration is engaged. The characteristics of Ca2+ handling in neurons of the substantia nigra pars compacta and the possible involvement of PD-related proteins in the control of Ca2+ homeostasis will be discussed in this review

Calcium and Endoplasmic Reticulum-Mitochondria Tethering in Neurodegeneration.

Mitochondria are key players of many physiological processes and deregulation of mitochondrial and/or mitochondria-related activity is unequivocally associated to numerous ageing-linked neurodegenerative disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Recently, the endoplasmic reticulum (ER) stress condition is emerging as a common feature relevant to the pathogenesis of this type of diseases. Mitochondria and ER are two compartments physically and functionally tightly interconnected and recent evidence revealed that the impairment in their communication might represent a common hit in different neurodegenerative diseases. ER-mitochondria contact sites are crucial for Ca2+ signaling since, upon the opening of ER Ca2+ release channels, microdomains of high [Ca2+] are generated in their proximity and Ca2+ can be taken up by the low-affinity mitochondrial uniporter. This transfer is essential in stimulated as well as in resting conditions to sustain cell metabolism and bioenergetics. Alterations in the ER-mitochondria juxtaposition are critical not only because they determine mitochondrial dysfunctions, but also because they compromise lipid metabolism, protein synthesis, and folding, thus demonstrating that the interaction between the two compartments is bi-functional. However, the functional consequences of these alterations on Ca2+ signaling and the possible involvement in the development of neurodegenerative conditions are currently largely unexplored. Here we will survey the recent literature in the field and discuss recent insights focusing on some cellular models expressing mutant proteins involved in the pathogenesis of familial forms of PD, AD, and ALS

Etiology and pathogenesis of Parkinson's disease: role of mitochondrial pathology

Neurons critically rely on mitochondrial activity: they are characterized by high energy demand and they are totally dependent on the process of oxidative phosphorylation to produce adenosine triphosphate. Thus, any impairment in mitochondrial function results in neuronal damage and degeneration. Some particular neuronal populations are more susceptible to mitochondrial damage, as it has been recently proposed for the ventral midbrain dopaminergic neurons, the degeneration of which represents a clinical sign of Parkinson\u2019s disease. Different cellular pathways are involved in the pathogenesis of this neurodegenerative disease, but intriguingly both sporadic and familial forms share common features that essentially recapitulate mitochondrial dysfunction. Mitochondrial biogenesis, bioenergetics, mitochondria dynamics, and quality-control process are the main affected pathways. General consensus agrees on the possibility that deficiency in these processes may represent the cause rather than the consequence of neurodegeneration. In this review, we will discuss these aspects and the substantial achievements that have been reached in recent years in identifying specific defects in precise biological processes, eg, mitochondrial quality control. The development of cell and animal genetic models has been an important tool to dissect numerous molecular details; for this reason, we will mainly refer to experiments performed on them

Calcium Pumps: Why So Many?

Ca2+\u2010ATPases (pumps) are key to the regulation of Ca2+ in eukaryotic cells: nine are known today, belonging to three multigene families. The three endo(sarco)plasmic reticulum (SERCA) and the four plasma membrane (PMCA) pumps have been known for decades, the two Secretory Pathway Ca2+ ATPase (SPCA) pumps have only become known recently. The number of pump isoforms is further increased by alternative splicing processes. The three pump types share the basic features of the catalytic mechanism, but differ in a number of properties related to tissue distribution, regulation, and role in the cellular homeostasis of Ca2+. The molecular understanding of the function of all pumps has received great impetus from the solution of the three\u2010dimensional (3D) structure of one of them, the SERCA pump. This landmark structural advance has been accompanied by the emergence and rapid expansion of the area of pump malfunction. Most of the pump defects described so far are genetic and produce subtler, often tissue and isoform specific, disturbances that affect individual components of the Ca2+\u2010controlling and/or processing machinery, compellingly indicating a specialized role for each Ca2+ pump type and/or isoform. \ua9 2012 American Physiological Society. Compr Physiol 2:1045\u20101060, 2012