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In vitro and ex vivo exploration of knock-in mouse models harboring mutations linked to late onset Parkinson’s disease Tatarnikov, Igor


Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting 2-3% of the population aged 65 or older. Most PD cases are idiopathic i.e. without identifiable cause. Currently, therapeutic options for PD, which include dopamine replacement and deep brain stimulation, manage motor symptoms relatively well immediately following diagnosis. Unfortunately, neither therapeutic strategy modifies the progression of the disease or address the plethora of non-motor symptoms. Historically, PD was thought of as a disease of the environment with little to no genetic contribution. However, within the last two decades mutations in Alpha-synuclein (SNCA), leucine-rich repeat kinase (LRRK2), vacuolar protein sorting 35 (VPS35), and DnaJ heat shock protein family (Hsp40) member C13 (DNAJC13) have been linked to autosomal dominantly inherited parkinsonism that is clinically indistinguishable from idiopathic PD. In this study we began by comparing mature primary cortical cultures derived from knock-in mice harboring either the Lrrk2 p.R1441C (RKI), or the Dnajc13 p.N855S (DKI) mutation. Previously we have shown that cortical neurons derived from Lrrk2 p.G2019S (GKI) mice showed an increased frequency of miniature excitatory post-synaptic currents (mEPSCs) with no change in amplitude. RKI neurons showed a significantly higher frequency of mEPSCs with no change in amplitude. DKI neurons were indistinguishable from their WT counterparts in the frequency or amplitude of mEPSCs. Next, we characterized the nigrostriatal dopamine system in 3-month-old DKI mice. By fast-scan cyclic voltammetry (FSCV) there were no significant changes in evoked release or clearance of dopamine. At a low dose, GBR-12909 acts as a potent and selective dopamine reuptake inhibitor. When applied to brain slices, DKI mutant animals were less responsive to dopamine active transporter (DAT) inhibition, suggesting lower levels of DAT, as verified by western blotting. Lastly, the effect of Lrrk2 kinase inhibition on the dopaminergic phenotypes previously described in Vps35 p.D620N (VKI) mice was examined. Lrrk2 kinase activity has been shown to be elevated in not only VKI mice but also in idiopathic PD. Previous work has shown changes in both DAT and VMAT2 levels along with increased evoked release and longer decays in 3-month-old VKI animals. Daily MLi-2 injections rescued the phenotypes previously observed.

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