This work of thesis was based on preliminary data obtained in the physiology laboratory of Università del Piemonte Orientale about alterations in the interaction between the endoplasmic reticulum and the mitochondria in immortalized astrocytes derived from a 3xTg Alzheimer’s mouse model. It has been known that a deregulation of ER-mitochondria Ca2+ transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of this transfer between the two organelles depends on the protein composition of the MERCS, which controls several key parameters which are altered in neurodegeneration. Concerning Alzheimer’s disease mouse model (3xTg-iAstro), the results obtained during the preliminary studies suggest that there is an overall increase short-distance mitochondrial-ER tethering, an activation of the ER-stress and reduction of protein synthesis, a mitochondrial and glycolytic energy metabolism impairment, an augmentation of intracellular ROS and intra-mitochondrial superoxide production and finally an impaired mitochondrial Ca2+ signaling as compared with WT-iAstro cells. Considering these assumptions, the aim of this work is to investigate the link between mitochondrial dysfunction, deregulation of Ca2+ homeostasis and dysproteostasis on iPSC-differentiated astrocytes from Parkinson’s disease patients with LRRK2(G2019S) mutation, using the SPLIC technique. This was possible thanks to the collaboration with the Achucarro Basque Center for Neuroscience, Bilbao, Spain, where all these experiments concerning the Parkinson’s disease took place. The preliminary results collected on this thesis suggest that there is an overall increase long-distance mitochondrial-ER tethering, an augmentation of protein synthesis. Moreover, a reduction of the basal Ca2+ level in the ER of ihA-PD and a consequent reduction in the calcium release from the ER, a reduction of the basal calcium level in the mitochondria and an increase in the level of cytosolic Ca2+ were described. All these data fit with the hypothesis of the role of the deregulation of ER-mitochondria Ca2+ transfer in pathophysiology of astrocytes, which contributes to the pathogenesis of neurodegenerative and other diseases.
I risultati esposti in questa tesi si basano su dati preliminari ottenuti nel laboratorio di fisiologia dell’Università del Piemonte Orientale, in merito ad alterazioni nell’interazione tra reticolo endoplasmatico e mitocondrio negli astrociti immortalizzati derivanti da modelli murini tripli transgenici Alzheimer (3xTg-AD). È ormai noto che una deregolazione del Ca2+ transfer tra reticolo e mitocondrio contribuisce alla patogenesi di malattie neurodegenerative e non solo. Gli effetti del trasferimento tra i due organelli dipendono dalla composizione proteica dei MERCS, i quali controllano diversi parametri che risultano essere alterati nella neurodegenerazione. In merito alla malattia di Alzheimer, i risultati di uno studio preliminare, ottenuti utilizzando il modello murino 3xTg-iAstro, suggeriscono che ci sia un aumento delle distanze short tra reticolo endoplasmatico e mitocondrio, un’attivazione dell’ER-stress e una riduzione della sintesi proteica, il danneggiamento del metabolismo energetico mitocondriale e glicolitico, un aumento della produzione intracellulare di ROS e di superossido intra-mitocondriale, infine un’alterazione del signalling di Ca2+ mitocondriale se confrontati con le cellule WT-iAstro (Dematteis et al, 2020; Tapella et al, 2022). Considerando questi presupposti, lo scopo di questo lavoro è stato quello di investigare il collegamento tra la disfunzione mitocondriale precedentemente osservata (Dematteis et al, 2020; Tapella et al, 2022)), la deregolazione dell’omeostasi del Ca2+ e la disproteostasi negli astrociti differenziati da iPSC di pazienti Parkinson con la mutazione LRRK2(G2019S), valutando una possibile implicazione dell’interazione tra i due organelli. Questo è stato possibile grazie alla collaborazione con l’Achucarro Basque Center For Neuroscience (Bilbao, Spain) dove tutti gli esperimenti riportati in questa tesi hanno avuto luogo. I risultati ottenuti suggeriscono che vi sia un significativo aumento delle distanze long tra reticolo e mitocondrio e un aumento della sintesi proteica. Inoltre, ho evidenziato una riduzione dei livelli basali di Ca2+ nel reticolo endoplasmatico e nel mitocondrio delle hiPSCs-PD e una conseguente riduzione del rilascio dello ione dal reticolo, e un aumento nei livelli basali di Ca2+ nel citosol. I dati qui riportati supportano l’ipotesi della deregolazione del Ca2+ transfer nella patofisiologia degli astrociti, che contribuisce alla patogenesi delle malattie neurodegenerative e molte altre.
Ruolo dell'interazione tra reticolo endoplasmatico e mitocondrio negli astrociti: un focus sulle malattie di Alzheimer e Parkinson.
PISTOLATO, BEATRICE
2021/2022
Abstract
This work of thesis was based on preliminary data obtained in the physiology laboratory of Università del Piemonte Orientale about alterations in the interaction between the endoplasmic reticulum and the mitochondria in immortalized astrocytes derived from a 3xTg Alzheimer’s mouse model. It has been known that a deregulation of ER-mitochondria Ca2+ transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of this transfer between the two organelles depends on the protein composition of the MERCS, which controls several key parameters which are altered in neurodegeneration. Concerning Alzheimer’s disease mouse model (3xTg-iAstro), the results obtained during the preliminary studies suggest that there is an overall increase short-distance mitochondrial-ER tethering, an activation of the ER-stress and reduction of protein synthesis, a mitochondrial and glycolytic energy metabolism impairment, an augmentation of intracellular ROS and intra-mitochondrial superoxide production and finally an impaired mitochondrial Ca2+ signaling as compared with WT-iAstro cells. Considering these assumptions, the aim of this work is to investigate the link between mitochondrial dysfunction, deregulation of Ca2+ homeostasis and dysproteostasis on iPSC-differentiated astrocytes from Parkinson’s disease patients with LRRK2(G2019S) mutation, using the SPLIC technique. This was possible thanks to the collaboration with the Achucarro Basque Center for Neuroscience, Bilbao, Spain, where all these experiments concerning the Parkinson’s disease took place. The preliminary results collected on this thesis suggest that there is an overall increase long-distance mitochondrial-ER tethering, an augmentation of protein synthesis. Moreover, a reduction of the basal Ca2+ level in the ER of ihA-PD and a consequent reduction in the calcium release from the ER, a reduction of the basal calcium level in the mitochondria and an increase in the level of cytosolic Ca2+ were described. All these data fit with the hypothesis of the role of the deregulation of ER-mitochondria Ca2+ transfer in pathophysiology of astrocytes, which contributes to the pathogenesis of neurodegenerative and other diseases.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15613