Altered motor phenotype and dopamine transmission associated with mutations of the parkinsonian gene LRRK2

The leucine-rich repeat kinase 2 mutation (LRRK2) G2019S in the kinase-domain is the most common genetic cause of late-onset autosomal dominant Parkinson’s Disease (PD), occurring in >85% of patients carrying this LRRK2 mutation. LRRK2-related PD is clinically indistinguishable from the classic idiopathic form, being characterized by classic neuropathological hallmarks such as progressive degeneration of the substantia nigra pars compacta (SNpc) dopaminergic neurons, gliosis and α-synuclein and ubiquitine-positive intraneuronal cytoplasmic inclusions. The main goal of this thesis work was to evaluate the role played by the kinase function of LRRK2 in the expression of motor phenotype and dopamine transmission in mice, since transgenic models reported so far failed to recapitulate the parkinsonian phenotype and its neuropathology. To directly explore the impact of the kinase-enhancing G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed. We enrolled two cohorts of G2019S knock-in (KI) mice and wild-type littermates (WT) and analyzed their motor activity at different ages (3, 6, 10, 15 and 19 months) using a set of complementary behavioral tests, specific for akinesia, bradykinesia and overall gait ability. Our study revealed that G2019S KI mice motor performance remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. To confirm that enhanced kinase activity accounts for this phenotype, we adopted a combined genetic and pharmacological approach. On one hand we performed a parallel longitudinal study in mice carrying a LRRK2 mutation (D1994S) that impairs kinase activity (kinase-dead, D1994S KD), on the other hand we administered two LRRK2 kinase inhibitors (H-1152 and Nov-LRRK2-11) in G2019S mice. We found that i) KD mice were not phenotypic and ii) LRRK2 inhibitors reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or in D1994S KD animals. In vivo LRRK2 targeting of kinase inhibitors was further substantiated by the reduction of LRRK2 phosphorylation at Ser935 in the striatum and/or cortex at efficacious doses of LRRK2 inhibitors. In order to investigate whether the hyperkinetic phenotype of G2019S mice was associated with dysfunction of striatal dopamine neurotransmission, we carried out a series of behavioral, biochemical, and neurochemical experiments. No changes in nigral dopamine cell counts or dopamine striatal density were observed in G2019S mice. However, the overall pattern of responses to a D2/D3 receptor agonist or antagonists and to D1/D5 receptor antagonists suggested an elevated tonic activation of dopamine receptors in G2019S KI mice. Furthermore, blockade of the dopamine transporter (DAT) resulted in an enhancement of motor performance of WT but not G2019S KI mice. Results from in vitro binding assays revealed a reduction in the DAT protein levels which was associated with an increased dopamine reuptake in G2019S KI mice. In vivo microdialysis showed a reduced metabolites/dopamine ratio in in the striatum of G2019S mice, suggesting a reduced dopamine turnover. Overall the data provide genetic and pharmacological evidence that the kinase activity of LRRK2 is highly implicated in the modulation of motor activity along with the striatal dopaminergic system. However, whether and how the observed changes in motor phenotype and dopamine transmission translate into the overt parkinsonian pathology remains a matter for speculation. It is also possible that G2019S KI mice reflect a pre-symptomatic stage of the disease, as observed in other genetic models of PD. Nonetheless, the present thesis work proposes G2019S KI mice as a valuable in vivo model to investigate the effects of LRRK2 inhibitors