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Unraveling the molecular mechanisms underlying alpha-synuclein oligomerization and cytotoxicity
Publication . Gonçalves, Susana Alexandra de Barros, 1983-; Outeiro, Tiago Fleming, 1976-
Neurodegenerative disorders (NDs) are proteinopathies characterized by the accumulation of misfolded and aggregated proteins. Either through loss of normal protein function and the generation of abnormal protein interactions, the protein network deteriorates inside neurons and subsequently along the neuronal networks. Parkinson’s disease (PD) is the second most frequent ND and is associated with the misfolding and aggregation of alpha-Synuclein (aSyn), a pre-synaptic protein whose function is still unclear. Importantly, aSyn dysregulation is also involved in other NDs, as Dementia with Lewy Bodies and Multiple System Atrophy, jointly referred to as Synucleinopathies. Thus, the study of aSyn became crucial for understanding the etiology of those pathologies. There is ample debate as to what the toxic species of aSyn are, although it has been postulated that misfolded oligomeric species of aSyn represent the toxic genus. This thesis aimed to generate new insights into the role of aSyn in health and disease, at a molecular level. To visualize aSyn in the biological orchestra of the cell, we first studied its intracellular dynamics in a cellular model through photoactivation microscopy. Using photoactivatable green fluorescent protein as a reporter, we found that the availability of the aSyn amino-terminus modulates its shuttling into the nucleus. This finding has important implications regarding both the species of aSyn that enter the nucleus and also the function of the protein within that compartment. aSyn was recently suggested to exist naturally as a tetramer. Due to the nuclear pore size, only monomeric or dimeric forms of aSyn can enter the nucleus, and this has been related to a deleterious effect and neurotoxicity, due to transcription deregulation. Interestingly, intracellular dynamics of aSyn was finely modulated by the HSP70 chaperone, PD-associated mutations and by the phosphorylation state of the protein on S129 site. We found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment. Also, A30P and A53T aSyn mutations increased the speed at which the protein moves between the nucleus and cytoplasm, respectively. Finally, specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. Importantly, a mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting that phosphorylation modulates aggregation, and thus, alter the normal aSyn intracellular dynamics. To better understand the aggregation process in disease, we focused on the initial steps of aSyn aggregation, thought to be the causative agents of pathology. We used cell-based models of Synucleinopathy to investigate the molecular mechanisms underlying aSyn oligomerization. In particular, we screened, in an unbiased manner, a subset of the human genome-wide collection of lentiviral RNA-interference constructs, targeting genes involved in signal transduction players, to identify modifiers of aSyn oligomerization, using the bimolecular fluorescence complementation assay (BiFC) as readout. Through this approach we identified 9 genetic modifiers of aSyn oligomerization. Interestingly, the hits we identified were functionally related, and associated with neuronal trafficking processes. We then characterized these hits with respect to their effects on aSyn aggregation, toxicity and protein levels. After this first level of general characterization, we further investigated the mechanism of action of the hits by assessing their effects on aSyn secretion, a central aspect in the spreading of aSyn pathology. aSyn is secreted under physiological conditions, via non-classical exocytosis, in association with exosomes, and possibly via other less conventional mechanisms. However, it was demonstrated that pathological and aggregated aSyn species can also be secreted, suggesting that aggregated and misfolded aSyn may be the key agent for propagation of aSyn pathology, possibly in a prion-like manner. Thus, in our study we selected four trafficking hits, based on the literature and on their relevance to secretory pathways. Ras-related Protein in Brain 8b (Rab8b), Rab11a, Rab13 and Synaptotagmin-Like Protein 5 were found to promote the clearance of aSyn inclusions and reduce aSyn toxicity. Moreover, we found that endocytic recycling and secretion of aSyn was enhanced upon expression of Rab11a or Rab13 in cells accumulating aSyn inclusions. Importantly, in cells with inclusions, the trafficking proteins co-localized with aSyn in inclusions. Altogether, our findings suggest specific trafficking steps may prove beneficial as targets for therapeutic intervention in Synucleinopathies, and should be further investigated in other models. Here, we also studied the effects of monoclonal aSyn antibodies on the early stages of aggregation using the BiFC assay. Our results support passive immunization against Synucleinopathies by demonstrating that extracellular administration of monoclonal antibodies can inhibit early steps in the aggregation process of aSyn. As aSyn seems to behave as a prion-like protein, immunization can be a mid-term strategy to delay the progression of Synucleinopathies. The present study uncovered novel aspects about the intracellular dynamics of aSyn and allowed the identification of new genetic players involved in the aggregation, toxicity, secretion and immunization of aSyn, opening novel avenues towards the understanding of the molecular bases of Synucleinopathies.
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Fundação para a Ciência e a Tecnologia
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SFRH
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SFRH/BD/79337/2011