Dissertation committee:
Βασιλακοπούλου Διδώ, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, Παν. Αθηνών
Δοξάκης Επαμεινώνδας, Ερευνητής Γ’, Ακαδημία Αθηνών
Ευθυμιόπουλος Σπυρίδων, Καθηγητής, Τμήμα Βιολογίας, Παν. Αθηνών
Ζαγοραίου Λασκαρώ, Ερευνήτρια Γ’, Ακαδημία Αθηνών
Παλικαράς Κων/νος, Επίκουρος Καθηγητής, Τμήμα Ιατρικής, Παν. Αθηνών
Παπαζαφείρη Παναγιώτα, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, Παν. Αθηνών
Τρουγκάκος Ιωάννης Π. , Καθηγητής, Τμήμα Βιολογίας, Παν. Αθηνών
Summary:
Parkinson's disease (PD) is the second most common neurodegenerative disorder, with unclear etiopathology, multifactorial profile, and substantial societal costs worldwide. There is currently no disease-modifying treatment. A-synuclein (SNCA) is an abundant neuronal protein normally localized in the presynaptic limbs and involved in the pathogenesis of PD, making it a top therapeutic target of interest. The significantly more frequent idiopathic forms of the disease suggest that an environmental stimulus, coupled with aging, is required for SNCA to oligomerize and localize to large protein inclusions, known as Lewy bodies, that will impact PD development. Among the causative proteins implicated in familial cases of neurodegeneration, those that orchestrate mass responses to environmental stress are likely to be at the core of neuropathogenetic mechanisms. These are the RNA-binding proteins (RBPs) that include, among others, T-cell intracellular antigen 1 (TIA1). TIA1 is a widely expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It includes domains that confer prion protein properties and is an essential protein responsible for stress granule formation in response to environmental stress. Its overexpression leads to misfolding of TAU in Alzheimer's disease, while its mutations lead to amyotrophic lateral sclerosis development. Based on the above, this Ph.D. thesis aimed to investigate whether TIA1 is involved or is capable of triggering PD pathogenesis. To answer the above questions, differentiated human neuroblastoma cell lines (SK-N-SH or SH-SY5Y) were used as a model system, while plasmid and viral vectors were used to modulate SNCA and TIA1 levels. Initially, the binding of TIA1 to SNCA mRNA was examined. After incubating different SNCA biotinylated probes with cell extracts, it was revealed that TIA1 selectively binds to the initial segment of the 3'-untranslated region (UTR) of SNCA and not to the 5'UTR or coding region. However, neither overexpression nor silencing of TIA1 altered endogenous SNCA mRNA or protein levels. Further, the co-immunoprecipitation analysis revealed no direct interaction between TIA1 and SNCA. In contrast, simultaneous overexpression of the two proteins resulted in a substantial increase in the aggregation and serine 129 phosphorylation of SNCA in both the soluble and insoluble fractions of the protein extracts. Proteasomes and autophagy pathway are essential in removing intracellular SNCA monomers and inclusions, respectively, while multiple kinases and phosphatases control SNCA phosphorylation. Further, SNCA levels are altered under the influence of mitochondrial oxidizing agents in both in vivo and in vitro conditions (e.g. neurotoxin MPP+, rotenone, 6-OHDA), and mitochondrial dysfunction drives SNCA accumulation, and mutations in genes related to mitochondrial metabolism lead to familial PD. Consequently, the role of TIA1 in these pathways was examined. TIA1 overexpression reduced total PP2A phosphatase activity in SH-SY5Y cells, but this effect was not due to the change in the levels of PP2A subunits or its intracellular regulators. Based on the preceding results and the fact that TIA1 is a sticky/prion protein, its interaction with the cell proteome was investigated next. Ascorbic acid peroxidase (APEX2) fused to TIA1 was used to label its interaction network in vivo, allowing subsequent ex vivo analysis. Using this method, all TIA1 partners at a distance of 20 nm under both normal and acute stress conditions were labeled. Then, using mass spectrometry for identification and co-immunoprecipitation for validation, the proteins directly associated with TIA1 were identified. Most proteins were associated with ribonucleoprotein complexes in both control and stress conditions. However, the repertoire of TIA1-protein partners was remarkably dissimilar between the two cellular conditions. Under physiological conditions, the TIA1-interacting proteome was enriched in cell-soluble processes associated with mRNA metabolism. Under stress conditions, TIA1-labeled partners displayed a broader subcellular distribution, including chromosomes and mitochondria. Taken together, these data highlight the spatial environment of TIA1 with its different partners in diverse cellular situations and pave the way for elucidating the role of TIA1 in these processes. In conclusion, robust data are presented on the effect of TIA1 on mechanisms linked to SNCA aggregation and phosphorylation as well as the proteome with which TIA1 interacts, facilitating the future investigation of the molecular mechanisms by which TIA1 exerts its effects.
Keywords:
a-synuclein, TIA1, Parkinson's disease, APEX2-TIA1,proximity labelling, proteasome, autophagy, mitochondria
