SIRT2 as a putative target in parkinson's disease therapy

Tese de mestrado, Biologia molecular e humana, 2008, Universidade de Lisboa, Faculdade de CiênciasResumo alargado em português disponível no documentoParkinson's disease is a progressive and fatal neurodegenerative disorders, affecting more than 1% of people over 65 years old. The disease is characterized by degeneration of dopaminergic neurons in the substantia nigra and by the presence of intracellular α-synuclein-containing inclusions known as Lewy bodies. The discovery of genes linked to rare familial forms of the disease as well as studies in in vitro and in vivo experimental models identified several putative targets for therapeutic intervention. Misfolding, oligomerization and aggregation of α-synuclein have been implicated in Parkinson's disease pathology. The sirtuins are a family of enzymes which regulate diverse cellular functions including metabolism, response to stress, and aging. Recently, sirtuins were found to be disease-modifiers in various models of neurodegeneration, including Parkinson's disease, where it prevents α-synuclein cytotoxicity and modulates its aggregation. In this study, our goal was to provide genetic validation of SIRT2 as a putative target for therapeutic intervention in Parkinson's disease. We started by identifying lentiviral vector encoded shRNAs in order to knock-down SIRT1, SIRT2, and SIRT3 in a mesencephalic mouse cell line (MES 23.5) which shows dopaminergic phenotypes. We found that the constructs selected lead to 80-90% knock-down of the desired targets as confirmed by real time PCR. Next, we were able to show that the levels of SIRT1 protein were also decreased, as expected. We then found that SIRT2 knock-down led to an increase in the acetylation of one of its known targets - α-tubulin. In addition, we investigated the effect SIRT2 RNAi on microtubule structure and also on the aggregation of a-synuclein, since one possibility is that SIRT2-based protection in Parkinson's models might be due to the modulation of these processes. Thus, this work establishes important tools for the validation of SIRT2 as a target, and opens novel avenues for therapeutic intervention