Abstract
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson’s disease. Here, we investigated whether the G2019S LRRK2 mutation causes morphological and/or functional changes at nigro-striatal dopamine neurons. Density of striatal dopaminergic terminals, nigral cell counts, tyrosine hydroxylase protein levels as well as exocytotic dopamine release measured in striatal synaptosomes, or striatal extracellular dopamine levels monitored by in vivo microdialysis were similar between ≥12-month-old G2019S knock-in mice and wild-type controls. In vivo striatal dopamine release was insensitive to the LRRK2 inhibitor Nov-LRRK2-11, and was elevated by the membrane dopamine transporter blocker GBR-12783. However, G2019S knock-in mice showed a blunted neurochemical and motor activation response to GBR-12783 compared to wild-type controls. Western blot and dopamine uptake analysis revealed an increase in dopamine transporter levels and activity in the striatum of 12-month-old G2019S KI mice. This phenotype correlated with a reduction in vesicular monoamine transporter 2 levels and an enhancement of vesicular dopamine uptake, which was consistent with greater resistance to reserpine-induced hypolocomotion. These changes were not observed in 3-month-old mice. Finally, Western blot analysis revealed no genotype difference in striatal levels of endogenous α-synuclein or α-synuclein bound to DOPAL (a toxic metabolite of dopamine). However, Serine129-phosphorylated α-synuclein levels were higher in 12-month-old G2019S knock-in mice. Immunohistochemistry confirmed this finding, also showing no genotype difference in 3-month-old mice. We conclude that the G2019S mutation causes progressive dysfunctions of dopamine transporters, along with Serine129-phosphorylated α-synuclein overload, at striatal dopaminergic terminals, which are not associated with dopamine homeostasis dysregulation or neuron loss but might contribute to intrinsic dopaminergic terminal vulnerability. We propose G2019S knock-in mice as a presymptomatic Parkinson’s disease model, useful to investigate the pathogenic interaction among genetics, aging, and internal or environmental factors leading to the disease.
Highlights
Autosomal-dominant missense mutations in the leucinerich repeat kinase 2 (LRRK2) gene (PARK8, OMIM 609007) cause familial late-onset Parkinson’s disease (PD) [59, 95]
We found that pSer1292 levels were ~8-fold higher in the striatum of 12-month-old G2019S KI mice compared to age-matched WT littermates (Fig. 1), indicating a clear-cut gain of kinase activity in the presence of the G2019S mutation
Striatal DA release is preserved in G2019S KI mice To investigate whether the exocytotic properties of DA terminals were affected by the G2019S mutation (Fig. 3), synaptosomes obtained from the striatum of 12-monthold mice were depolarized with a sequence of three 90-s pulses (18 min away) of 10 mM or 20 mM K+ (Fig. 3a)
Summary
Autosomal-dominant missense mutations in the leucinerich repeat kinase 2 (LRRK2) gene (PARK8, OMIM 609007) cause familial late-onset Parkinson’s disease (PD) [59, 95]. LRRK2 mutations occur in 1–2% of sporadic cases [28, 70] and recent genome wide association studies (GWAS) showed that common variations in the LRRK2 locus increase the risk of disease, pointing to a crucial role of LRRK2 in the pathogenesis of PD. The cellular activity of LRRK2, probed with antiautophosphorylation antibodies against Serine 1292 [67, 72] and by measuring the phosphorylation of a subset of Rab GTPase which are bona fide LRRK2 cellular substrates [76], revealed a homogeneous increase of LRRK2 kinase activity in the presence of pathogenic mutations, which is not limited to the G2019S mutant as it occurs in vitro
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