Ottolini, Denis
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 [Tesi di dottorato]

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.

In relazione con
BIO/10 - Biochimica

Tesi di dottorato. | Lingua: | Paese: | BID: TD13053718