Polyglutamine diseases (PolyQ): construction and characterization of yeast models

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

There are a number of serious diseases which have in common the inappropriate folding of a particular protein, as polyglutamine disease group. Machado-Joseph‟s and Huntington‟s disease are grouped in the polyglutamine diseases, being the cause of the disease the presence of a CAG repeat tract in the causative gene, ATXN3 and HTT, respectively. This CAG repeats tract leads to the formation of polyglutamine tract in ataxin-3 and huntingtin proteins, respectively, that do not allow the correct protein folding. In diverse models, the expansion of the poluglutamine tract into disease related-proteins, promote their aggregation and their negative effect on cellular metabolism were described as associated to toxic effect. In this study, it was proposed the construction of the first yeast model for Machado-Joseph‟s disease and the characterization of the role of autophagy in the yeast model for Huntington‟s disease during yeast life cycle. For the yeast model for Machado-Joseph‟s disease we successfully constructed a Tet On system harboring the variant 1 genes, normal and expanded, fused with GFP gene, to further allow the subcellular localization of the expressed protein into yeast cells. In yeast model for Huntington‟s disease the chronological lifespan (CLS) of yeast cells at different physiological states after expressing the normal and the pathogenic protein and their subcellular localization, using epifluorescent microscopy was studied. It was demonstrated that cells harboring the huntingtin with 103 glutamines (pathogenic protein) during the inhibition of autophagic process, by chloroquine, presents an increased in CLS and the formation of large foci that could correspond to aggregosomes. In aged cells this effect is more pronounced leading to the hypothesis that in these cells the autophagic mechanism is impaired and enhances the toxic effects of expressed huntingtin 103Q and that the inhibition of the autophagic process can rescue the cells from the toxic effects. On the other hand, when the normal huntingtin (25Q) was expressed at exponential phase the inhibition of autophagy led to a drastic decrease on CLS, demonstrated that in functional cells the autophagy had a positive effect being a crucial process, probably removing unwanted intracellular components and providing energy.