DECIPHERING THE ROLE OF THE SERINE THREONINE KINASE LRRK2 IN THE CENTRAL NERVOUS SYSTEM AND IN PERIPHERAL TISSUES: IMPLICATION FOR PARKINSON\ubfS DISEASE TREATMENT

Leucine-rich repeat kinase 2 (LRRK2) encodes a large and complex protein which is widely expressed in brain and peripheral organs. Mutations in LRRK2 are a major cause of inherited and sporadic Parkinson\u2019s disease (PD), an age-dependent neurodegenerative disorder characterized by neuronal damage in multiple brain regions and consequent motor defects. Studies performed in the Central Nervous System (CNS) support a role of the protein in different functions, but despite extensive studies, the pathophysiological role of LRRK2 still remains enigmatic. LRRK2 encompasses two enzymatic domains (a kinase and GTPase domain) and multiple protein-protein interaction modules that can scaffold several protein complexes. Therefore, defining the LRRK2 interactome and understanding how its composition is modulated can be useful to identify its function. Since LRRK2 is notably involved in vesicular trafficking, the first aim for the proposed study was to investigate the role of the N-terminal domain of LRRK2 as a scaffold and its functional impact on synaptic vesicle (SV) dynamics in the N2a neuroblastoma cell line. High resolution fluorescence microscopy techniques coupled to genetically encoded sensors of SV fusion and recycling, demonstrated that the N-terminal domain is crucial for LRRK2 targeting to membrane structures and for its interaction with proteins involved in the control of SV mobilization and recycling. Our data confirm LRRK2 as a component of the presynaptic protein network, coordinating both storage and mobilization of SV through its N-terminal domain. Mutations in this domain (PD-linked E193K variant studied in this work) or modulation of its phosphorylation (in particular at serine 935) affects LRRK2 function via perturbation of its physiological network of interactors, thus resulting in impaired SV dynamics (Chapter I). In addition to being expressed in the CNS, LRRK2 can be detected in a variety of peripheral organs, including the endocrine pancreas, thus leading to the suggestion that the protein may be implicated in other functions. Therefore, in the second part of the project we focused on the endocrine pancreas and explored the possible involvement of LRRK2 in the control of insulin release and glucose homeostasis, using both cellular and animal models. Through pharmacological, molecular, and functional approaches we demonstrated that LRRK2 and its kinase activity control the glucose-stimulated insulin secretion, by modulating the trafficking of insulin granules. Expression of PD-associated LRRK2 mutants, characterized by enhanced kinase-activity, increased basal insulin release, and decreased the glucose-stimulated secretion, thus affecting the animal\u2019s metabolic profile. These findings identify a new role of LRRK2 in the control of glucose homeostasis and support the idea that LRRK2 may contribute to PD development and/or progression also indirectly through modulation of glucose homeostasis and cellular energetics. Not surprisingly, drugs used in type 2 diabetes and intended to restore the glucose homeostasis are among the most promising treatments currently being considered as a possible new therapy for PD (Chapter II). In the complex, our data indicate LRRK2 as a master regulator of secretory vesicles trafficking in both neuronal and endocrine cells of the pancreas and suggest the protein as important therapeutic target in multiple diseases