Intracytoplasmic protein aggregates called Lewy Bodies (LB) characterize the neurodegeneration in Parkinson’s Disease (PD). However, whether aggregates are linked to cell death or they represent a protective mechanism is debated. Recent studies suggest that autophagy participates in the degradation of cytoplasmic protein inclusions. Moreover, growing evidence shows that mitochondrial impairments and altered mitochondrial fission and fusion play a role in the pathogenesis of PD. Synphilin-1 is a protein that is present in LB and that interacts with key elements of PD pathogenesis, such as α-synuclein. Overexpression of synphilin-1 in various cellular models leads to cytoplasmatic inclusions that fulfil the criteria of aggresomes, dynamic structures formed under proteolytic stress. HtrA2/Omi is a mitochondrial protease that is thought to be involved in protection of mitochondria against stress in physiological conditions, besides its pro-apoptotic function under apoptosis induction. Loss of HtrA2/Omi function leads to neurodegeneration in mouse models. Mutations in both synphilin-1 and HtrA2/Omi have been found in PD patients. We used HEK293 cells overexpressing wild type (WT) or R621C mutant synphilin-1 to evaluate if autophagy activation may influence cell viability by modulating synphilin-1 inclusions. Moreover, we studied also the effects synphilin-1-overexpression and aggregation on mitochondria. In addition, we evaluated the consequences of loss of HtrA2/Omi on mitochondrial function and morphology in fibroblasts from knockout mice, as well as in HeLa cells and in Drosophila melanogaster S2R+ cells. We observed co-localization of synphilin-1 inclusions with autophagic structures and the pharmacological activation of autophagy was effective in reducing the percentage of cells bearing synphilin-1 inclusions. However, this treatment couldn’t protect the cells from apoptosis induced by proteasome inhibition. Synphilin-1-overexpressing cells revealed higher levels of mitochondrial reactive oxygen species (ROS) and reduced mitochondrial membrane potential (MMP) compared to empty vector controls. HtrA2/Omi deficiency caused accumulation of ROS within mitochondria and reduced MMP as well, together with morphological alterations of the mitochondria. Interestingly, we observed a selective up-regulation of the fusion factor OPA1 in cells lacking HtrA2/Omi and co-immunoprecipitation experiments showed that these two proteins can physically interact. To conclude, our findings support the view that aggresomes are actively built to remove excesses of noxious proteins; however, the enhancement of their clearance via autophagy is not sufficient to protect against proteasome inhibition and mitochondrial impairments in the presence of high levels of aggregate-prone proteins. Concerning HtrA2/Omi, we confirmed previous reports showing an involvement of this protease in maintaining mitochondrial homeostasis and we reported for the first time a direct effect of loss of HtrA2/Omi on mitochondrial morphology. Finally, we showed a novel role of HtrA2/Omi in the modulation of OPA1.
(2010). Cell-based analysis of dynamic aspects of molecular mechanism involved in the pathogenesis of Parkinson's disease. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).
|Data di pubblicazione:||29-mar-2010|
|Titolo:||Cell-based analysis of dynamic aspects of molecular mechanism involved in the pathogenesis of Parkinson's disease|
|Settore Scientifico Disciplinare:||MED/04 - PATOLOGIA GENERALE|
|Scuola di dottorato:||Scuola di Dottorato in Medicina Traslazionale e Molecolare|
|Corso di dottorato:||MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 45R|
|Citazione:||(2010). Cell-based analysis of dynamic aspects of molecular mechanism involved in the pathogenesis of Parkinson's disease. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).|
|Parole Chiave:||Synphilin-1; HtrA2/Omi; aggregation; autophagy; apoptosis; fusion; mitochondria|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|