Analisi del ruolo fisiopatologico della proteina LRRK2,responsabile della malattia di Parkinson familiare di tipo 8

Parkinson’s disease (PD) is the second most common neurodegenerative disease, characterized by selective loss of dopaminergic neurons in thesubstantia nigra. Mutations in the gene coding for Leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinson’s disease (PARK8). LRRK2 is a large multidomain protein of 2527 amino acids composed of different functional domains. The pathological mutations have been associated with increase of LRRK2 kinase activity, although very little is known about its physiological substrates and its role in PD pathogenesis. In this work, using a GST-pull down approach coupled to mass spectrometry, I have identified a putative new interacting protein of LRRK2, the component of exocyst complex Sec8. To examine this possibility, I performed co-immunoprecipitation experiments andin vitrokinase assay to investigate the ability of wild type or mutant LRRK2 to phosphorylate Sec8. I was able to observe a physical interaction between Sec8 and LRRK2 in transfected neuronal cells. Moreover LRRK2 is able to phosphorylate Sec8 bothin vitroandin vivo. During my research work I have also investigated the LRRK2 subcellular localization. Using neuronal transfected cell, I found that LRRK2 is associated with outer membrane of mitochondria but is unable to enter into the mitochondria even if we fuse LRRK2 with matrix or intermembrane space localization signals.