Unit:
Διατμηματικό / Διϊδρυτικό ΠΜΣ Μοριακή ΙατρικήLibrary of the School of Health Sciences
Supervisors info:
Ευθυμιόπουλος Σπύρος, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Βεκρέλλης Κώστας, Ερευνητής Β, Τομέας Βασικής Έρευνας, ΙΙΒΕΑΑ
Πολίτης Παναγιώτης, Ερευνητής Γ, Τομέας Βασικής Έρευνας, ΙΙΒΕΑΑ
Original Title:
Μελέτη της επίδρασης αναστολέων της φωσφολιπάσης Α2 στην τοξικότητα και την φλεμονώδη απόκριση που προκαλεί η συσσώρευση της α-συνουκλεΐνης in vitro και in vivo
Translated title:
Effect of PLA2 inhibitors in α-synuclein mediated toxicity and inflammation in vitro and in vivo
Summary:
α-Synuclein is a presynaptic protein that is linked with the pathogenesis of Parkinson’s Disease (PD). Under yet unknown circumstances α-synuclein accumulates and forms oligomeric species that are considered to be responsible for neuronal toxicity. Polyunsaturated fatty acids (PUFAs) interact with α-synuclein in a manner that promotes its aggregation. Fatty acids, such as arachidonic acid, are released from membrane phospholipids by phospholipase A2 (PLA2). Importantly, elevated activity of PLA2 and extensive release of membrane PUFAs have been observed in PD. Increased PAL2 activity may also increase arachidonic acid release and its subsequent metabolism to eicosanoids. In fact, eicosanoid signaling plays a major role in inflammatory processes, which have been proposed to contribute to PD pathology.
The aim of this study was to evaluate the effect of PLA2 inhibition and subsequent reduction of arachidonic acid in α-synuclein mediated toxicity in vitro and in the progression of inflammatory processes in vivo. To address these questions, we have applied novel PLA2 inhibitors on SH-SY5Y neuroblastoma cell line inducibly overexpressing wild type α-synuclein. We have found that certain PLA2 inhibitors increase the viability of neuronally differentiated cells overexpressing α-synuclein vs control cells expressing the basal levels of the protein. Further investigation indicated that increased cell survival was accompanied by a robust reduction of intracellular monomeric and oligomeric α-synuclein levels. To dissect how the inhibitors induce the reduction in α-synuclein levels, we tested the effects of the compounds in the secretory and degradation pathways of the protein. Quantification of extracellular α-synuclein in the conditioned medium of the cells indicated no changes in the levels of secreted α-synuclein following application of the PLA2 inhibitors. Assessment of the proteasomal activity using a specific fluorogenic substrate showed no significant changes upon PLA2 inhibition. Macroautophagy was tested by the formation of autophagic vesicles using the LC3 marker. Our results indicated that the inhibitors failed to induce any changes in the autophagic flux. Furthermore, the evaluation of the CMA pathway was assessed using SH-SY5Y cells expressing mutant ΔDQ α-synuclein which is not recognized by the lysosomal receptor and thus is not degraded by the CMA pathway. ΔDQ α-synuclein was also dramatically reduced upon PLA2 inhibition suggesting no particular involvement of the CMA pathway.
In order to study the effect of PLA2 inhibition in vivo we used transgenic mice that overexpress the human mutant A53T α-synuclein. This model is characterized by a three-fold increase of α-synuclein in the brain, which results in an increase in activated microglia and astrocytes suggestive of an established inflammatory response. The most potent inhibitor was intraperitoneally administred in transgenic mice and their wild type littermates in order to evaluate how PLA2 inhibition affects the levels of inflammatory mediators and α-synuclein. Intrestingly, α-synuclein levels in the cerebral cortex were significantly reduced, approaching those of wild-type mice. In addition, assessment of the populations of astrocytes, microglia and macrophages in the brain using flow cytometry revealed a small decrease in relation to those of non-treated mice. Finally, we tested the levels of the inflammation - related cytokines IFN-γ and IL-10. We found that the cytokines levels were significantly altered in the spleen and lymphnodes of both wild-type and transgenic mice upon treatment. However, there were no significant changes in the cytokine levels in the brain.
These results suggest a potential role of PUFAs in the biochemical properties of α-synuclein. PLA2 inhibition may promote changes in α-synuclein conformation resulting in increased clearance and reduced oligomer formation of the protein. The fact that α-synuclein levels were also reduced in mouse brain following PLA2 inhibition is probably suggestive of a common arachidonic acid-dependent mechanism responsible for α-synuclein clearance. Our in vivo results showing mild changes in the levels of inflammatory cells and their mediators are not yet conclusive. As such, further investigation is still required in order to establish the role of PLA2 inhibition in a-synuclein-related inflammation. Dissecting the link between lipid association and α-synuclein clearance could reveal new directions in approaching synucleinopathies and other amyloidogenic diseases.
Keywords:
α-synuclein, Phospholipase A2, Lipids, Toxicity, Inflammation
Number of references:
140
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XYLAKI MARY thesis.pdf
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