Mitochondrial targeting in LRRK2-associated parkinsonism (PARK8)

Among monogenic forms of Parkinson's disease (PD), the most frequent is associated with the leucine-rich repeat kinase2 (LRRK2) mutation, named PARK8 and the most common G2019S mutation. LRRK2 is expressed in the dopamine (DA) neurons of the substantia nigra compacta, suggesting a role in PD degeneration. In PD, mitochondrial complex I is impaired thus inhibitors of this complex and 6-OHDA are used to lesion the DA nigrostriatal system and induce experimental parkisonism. To study the role of LRRK2, models carrying LRRK2 pathogenic mutations have been developed. LRRK2 mutants cause neurodegeneration impairing mitochondrial activity indicating a prominent role of this structure in LRRK2-mediated death signals. Kinase regulation of mitochondrial dynamics are pivotal in these processes in relation to PD onset and expression of G2019S mutation alters the homeostasis of mitochondrial Ca2+. Drugs able to correct LRRK2-associated mitochondrial dysfunction may attenuate parkinsonism in mice. Aim 1. We will investigate whether the G2019S mutation renders dopamine (DA) neurons more prone to death over aging or following exposure to parkinsonian toxins, ultimately making mice more susceptible to develop parkinsonism. Therefore, our first main goal is to provide behavioral, electrophysiological (striatal slices), biochemical (cell lines and mesencephalic/cerebrocortical neurons) and neurochemical (microdyalisis/synaptosomes) characterization of i) mice over-expressing human LRRK2 carrying the G2019S mutation; ii) LRRK2 Knock-Out (KO) mice; iii) LRKK2 G2019S Knock-In (KI) mice and iv) mice expressing a LRRK2 mutation (D1994S) associated with null kinase activity (Kinase Dead; KD mice). Aim 2. We will take advantage of these multiple genetic models and expertises available, to evaluate: i) the relation between mouse phenotype and age (3, 6 and 12 months); ii) the effect of exposure to various parkinsonian toxins such as MPTP, 6-OHDA and rotenone. Aim 3. In addition, mitochondrial drugs will be administered to correct behavioral and biochemical deficits in LRKK2 mice in vivo and ex vivo, as well as in cell lines. In particular, drugs specific for mitochondrial targets (permeability transition pore, radical oxygen production, dynamin-related GTPases) will be used in order to protect from in vitro and in vivo degeneration DA neurons carrying the characterized LRRK2 mutations.