|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Generazione di cellule staminali pluripotenti indotte mutate in TBK1 per la caratterizzazione e la correzione della patologia della SLA|
|Titolo in inglese||Generation of novel TBK1-mutant hiPSCs for the characterization and correction of ALS pathology|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||CHIMICA E TECNOLOGIA FARMACEUTICHE (D.M. 270/04)|
|Data inizio appello||2018-11-08|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
La sclerosi laterale amiotrofica (SLA) è una malattia neurodegenerativa ad insorgenza adulta, caratterizzata dal punto di vista clinico dalla progressiva degenerazione dei motoneuroni superiori ed inferiori. Alterazioni in diversi geni sono state correlate a SLA ma le esatte cause che portano a degenerazione dei motoneuroni sono ancora sconosciute. Inoltre, ad oggi, non è ancora stata identificata una terapia efficace.
Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease, clinically characterized by the progressive degeneration of upper and lower motoneurons. Alterations in several genes have been correlated with ALS, but the exact causes leading to motoneuron degeneration are still unknown. Moreover, up to date no effective therapy has been found. Mutations in TANK-binding kinase 1 (TBK1) have been only recently linked to ALS and still very little is known about its contribution to ALS pathogenesis. In this thesis, we aimed to investigate the pathophysiological alterations associated with mutations in TBK1 gene in order to characterize and to rescue the detrimental ALS phenotype. To do this, we generated hiPSCs from one ALS patient harboring a 358+2T>C loss-of-function mutation in TBK1 gene, and differentiated them into motoneurons. Using this in-vitro model, we found that mTBK1 motoneurons are characterized by TBK1 haploinsufficiency and reduced activation of the autophagic receptor p62, which as a consequence accumulates in its unphosphorylated form into aberrant cytosolic aggresomes. Moreover, we observed increased neuronal loss in cultured mutant motoneurons when compared to healthy control and, accordingly, alterations in mitochondrial homeostasis and increased levels of pro-apoptotic proteins. Interestingly, mutations in TBK1 lead also to a dramatic alteration of the molecular composition of excitatory synapses, suggesting a detrimentally decreased neuronal activity. Finally, based on the dramatic alterations characterizing mutant motoneurons, we screened for a small-molecule library of ion-channels ligands in order to rescue the ALS-related phenotypes. We found that two known, FDA-approved Ca2+ blockers, Nimodipine and Amiodarone, reduce the load of p62-positive protein aggregates within mTBK1 MNs. These last findings are particularly noteworthy because they show that the regulation of Ca2+ channels might be a promising therapeutic strategy in ALS.