Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Author:
Leandrou Emmanouela
Dissertation committee:
Γεώργιος Παρασκευάς, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ, Επιβλέπων
Κώστας Βεκρέλλης, Ερευνητής Α’, Κέντρο Βασικής Έρευνας, Ίδρυμα Ιατροβιολογικών Ερευνών Ακαδημίας Αθηνών
Ευαγγελία Εμμανουηλίδου, Επίκουρη Καθηγήτρια, Τμήμα Χημείας, ΕΚΠΑ
Λεωνίδας Στεφανής, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Παναγιώτα Παπαζαφείρη, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Αγγελική Χρόνη, Ερευνήτρια Α΄, Ινστιτούτο Βιοεπιστημών και Εφαρμογών, ΕΚΕΦΕ «Δημόκριτος»
Δήμητρα Θωμαΐδου, Ερευνήτρια Α΄, Τμήμα Νευροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Original Title:
Διερεύνηση μηχανισμών μετάδοσης της παθολογίας της α-συνουκλεΐνης σε μοντέλα παρκινσονισμού
Translated title:
Investigation of the mechanisms involved in α-synuclein transfer of pathology in Parkinson’s Disease models
Summary:
α-synuclein, a small pre-synaptic neuronal protein, is both biochemically and genetically linked with Parkinson’s Disease (PD) pathogenesis. α-synuclein was considered an exclusively cytoplasmic protein, until several studies confirmed its presence in the extracellular space. However, the mechanism of α-synuclein release is still unclear either under normal or pathological conditions. The levels of extracellular α-synuclein is of great importance since it has been implicated in the cell-to-cell propagation of pathology between interconnected brain areas, a prominent characteristic of the PD brain.
The first part of this thesis focused on the identification and pharmacological targeting of the specific Voltage-gated Calcium Channels (VGCCs) that mediate α-synuclein release both in vitro and in vivo. Initially, the mechanism of α-synuclein secretion was studied in vitro by application of different selective VGCCs blockers into mouse embryonic cortical neurons and quantification of secreted α-synuclein by an in house ELISA. The functionality of the blockers was confirmed by live calcium imaging in cortical neurons. In vivo, α-synuclein release was studied in transgenic mice expressing the human A53T mutant form of α-synuclein. In these mice, the involvement of each VGCC type in α-synuclein secretion was assessed by local administration of each specific VGCC blocker by in vivo reverse microdialysis. The results obtained from this study indicate that α-synuclein secretion in vitro and in vivo is a VGCC-dependent process, which in mouse striatum, is modulated, at least in part, by the N-type VGCCs, whereas the L and P/Q-type VGCCs are not probably involved.
α-synuclein secretion is a physiological process in neurons. However, abnormal α-synuclein secretion could promote its aggregation, which has been linked with sustained neuroinflammation in PD, aggravating neuronal degeneration. Even though α-synuclein can be phagocytosed by microglia and/or astrocytes, the molecular pathways that trigger and prolong inflammation in the PD brain remain unclear.
In the second part of the thesis, the human α-synuclein A53T transgenic mouse model was used to investigate α-synuclein derived neuroinflammation. This model expresses high levels of both oligomeric and monomeric α-synuclein and exhibits sustained inflammatory responses as indicated by significant elevations in the levels of endogenous IgG antibodies and pro-inflammatory cytokines (TNFα, IFNγ and IL1β). In vitro experiments on primary microglial cells indicated that the oligomeric, but not monomeric, α-synuclein conformers were able to activate microglia as shown by NF-κB activity and increased TNFα release. Further characterization of the inflammatory environment in A53T striatum by using 3D cell reconstruction and morphometric analysis of the GFAP+ astrocytes revealed increased number and distinct morphological alterations in the A53T mice compared to wild type mice. Subsequent analysis of the striatum of A53T mice using immunofluorescence and immunoblotting revealed an activation of the p38MAPK pathway in microglia that stimulated the NF-κB pathway in astrocytes.
Aberrant astrocytic calcium signaling has been linked with the pathogenesis of several neurodegenerative diseases and could contribute to the initiation or sustenance of neuroinflammation. To assess astrocytic calcium signaling, fluorescence measurements in individual GFAP+ astrocytes were performed. This approach revealed a significant increase in the levels of Cav3.2 T-type VGCCs in the striatum of A53T mouse compared with wt littermates. To investigate the molecular link between inflammatory responses and Cav3.2 induction, mouse primary quiescent astrocytes were treated with cytokines and Cav3.2 was assessed by immunoblotting. These experiments showed that TNFα and IL-1β can potently induce functional Cav3.2 channels in a NF-κB-dependent manner. Secretomic analysis in quiescent astrocytes overexpressing the Cav3.2 VGCCs highlighted the secretion of Insulin-like growth factor-binding protein-like 1 (IGFBPL1), a potent regulator of neuronal axonal growth.
Overall, the identification of a specific type of VGCC as a regulator of α-synuclein secretion set the grounds for the selective pharmacological targeting of this channel to control the levels of secreted α-synuclein. Increased expression of α-synuclein could result in elevated extracellular levels of the protein which in turn promotes the accumulation of aberrant oligomeric species inducing neuroinflammatory responses. The data provided here suggest that the elevation of astrocytic Cav3.2 VGCCs could act as a compensatory mechanism that protect neurons from α-synuclein-induced inflammation via boosting IGF1 signalling.
Main subject category:
Health Sciences
Keywords:
Parkinson's Disease, Alpha-synuclein, Calcium signalling, Neuroinflammation
Number of references:
505
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Emmanouela_Leandrou_PhD.pdf
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