Διερεύνηση του μηχανισμού δράσης της εκκρινόμενης ασυνουκλεΐνης: επιπτώσεις στην παθογένεια και στην εξέλιξη της νόσου Πάρκινσον ; Investigation of the Mechanism of Action of secreted αsynuclein: effects on pathogenesis and progression of Parkinson's disease

In this thesis we studied the secretion mechanisms of alpha-synuclein and its effect on cell homeostasis. The study was performed by using proliferating SH-SY5Y neuroblastoma cell lines inducible expressing wild type (WT) α-synuclein. Τhe effect of chemical compounds, that inhibit specific non-classical pathways of secretion, showed that a-synuclein secretion is dependent on changes of intracellular calcium concentration. Since alpha-synuclein has been shown to be partly released by exosomes, we also attempted to examine the mechanism involved in exosome internalization by cells and thus the effect of a-synuclein as an exosomal cargo. In order to study the uptake mechanism, we initially examined the cells’ ability to internalize exosomes. The cells used were proliferating and differentiated neuroblastoma SH-SY5Y cells and primary neurons. Staining of the isolated exosomes with the specific dye Bodipy-Tr-Ceramide, and their subsequent application on the different cell cultures, showed that exosomes are readily internalized by the cells. Exosome uptake by cells was through a combination of different endocytosis mechanisms, but this result needs further elucidation. In addition, we attempted to study the effect of a-synuclein as exosomal cargo on neuronal homeostasis. The cell morphological parameter that was examined was cell volume, which was measured after the application of the different types of a-synuclein containing exosomes. Our results showed that mutant (A53T) protein may cause the reduction of cell volume. Finally, by using SH-SY5Y inducible expressing mutant asynuclein (ΔDQWT) that cannot be degraded by the lysosome, we examined the effect of chaperone mediated autophagy (CMA) deficiency on a-synuclein secretion either as free or associated with exosomes. Our study showed that a-synuclein secretion is reduced significantly in cells expressing the mutant form of the protein when compared with control cells expressing the wild type form. Intracellular protein levels did not vary. In addition, we observed ...