Please use this identifier to cite or link to this item: https://hdl.handle.net/10316/41347
Title: Protein misfolding and aggregation in neurodegenerative disorders
Authors: Afonso, Yolanda Sofia Pires 
Orientador: Domingos, Pedro
Girão, Henrique Manuel Paixão dos Santos
Keywords: Doenças neurodegenerativas; Proteínas
Issue Date: May-2016
Abstract: Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Parkinson’s disease (PD) and Huntington Disease (HD). The misfolding and aggregation of specific proteins contributes to neuronal degeneration in somewhat specific areas of the brain. However, the mechanisms by which these proteins lead to degeneration remains poorly understood. The main goals of this study was to elucidate and identify the molecular determinants of the subcellular localization of alpha-synuclein (α-syn) in PD and also to study the effect of N-terminal phosphorylation in the aggregation and toxicity of mutant huntingtin (Htt) in HD using Drosophila melanogaster as model. Previous results from our lab have shown that the mutant form of α-syn protein (α-syn-EGFP A30P) fails to localize the photoreceptor’s synapses. In this study we used RNAi knockdown screen to identify gene modulators of α-syn-EGFP A30P localization by performing head cryossections of adult flies using GMR-Gal4 driver in order to express the protein α-syn (WT and mutant form) in the Drosophila eye. We validated that α-syn-EGFP A30P has a mislocalized distribution throughout the photoreceptors cytoplasm. Using specific genetic knockdown for spaghetti squash, tomosyn isoform C and synaptotagmin 4, we saw an increase of α-syn-EGFP A30P localization at the synapse and these specific interactors may be possible modulators of this localization. Phosphorylation pathways have been shown to modulate the toxicity of mutant Htt in vitro by affecting its oligomerization and aggregation dynamics. We used a Drosophila model where the amino acid residues T3, S13 and S16 of Httex1 97Q were mutated to aspartate (T3D, S13D and S16D - phosphomimics) or alanine (T3A, S13A and S16A - phosphoresistant). We did functional assays to evaluate the effect of N-terminal phosphorylation on motor abilities and survival rate and we also performed dissections of larval eye-imaginal discs and adult brains in order to characterize the pattern of aggregation of these mutants. Moreover, we studied the effect on Httex1 protein aggregation of particular phosphatases that may dephosphorylate NT17 using RNAi gene knockdown experiments in dopaminergic neurons, using TH-Gal4 driver. From our results it was possible to confirm that single phosphorylation on NT17 domain modulates Htt aggregation and neurotoxicity in Drosophila since phosphomimic mutants showed an improvement in motor function with the exception of the T3D mutant. Phosphomimic mutants also exhibited increased life span compared to the phosphoresistant mutants. Interestingly, phosphomimic mutants showed bigger aggregates than phosphoresistant mutants in two different cell types analyzed, the dopaminergic neurons of the adult brain and the photoreceptor cells of the larval eye-imaginal discs. We also test the effect of genetic knockdown for specific protein phosphatases on dopaminergic neurons and a significant reduction of mutant Htt aggregation was observed when PP1α-96A and PP1-87B protein phosphatases where downregulated. These findings suggest the mislocalization of α-syn-EGFP A30P may due to its interaction with other proteins in the cell and impairs its traffic to the synapse. In HD, phosphorylation has an important role in modulating aggregation and toxicity of Httex1 and specific phosphatases may improve the outcomes.
Description: Dissertação de mestrado em Investigação Biomédica, apresentada à Faculdade de Medicina da Universidade de Coimbra
URI: https://hdl.handle.net/10316/41347
Rights: openAccess
Appears in Collections:UC - Dissertações de Mestrado
FMUC Medicina - Teses de Mestrado

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