title: Age-dependent dopamine transporter dysfunction and Serine129 phospho-α-synuclein overload in G2019S LRRK2 mice.
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.
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Longo
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Mercatelli
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Novello
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Ludovico
authors.initials: L
authors.lastname: Arcuri
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Brugnoli
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Vincenzi
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Russo
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Berti
authors.affiliation: Departments of Pharmacology and Chemistry, University of Michigan, 930 North University, Ann Arbor, MI, 48109, USA.
authors.forename: Omar S
authors.initials: OS
authors.lastname: Mabrouk
authors.affiliation: Departments of Pharmacology and Chemistry, University of Michigan, 930 North University, Ann Arbor, MI, 48109, USA.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
authors.affiliation: Department of Autoimmunity/Transplantation/Inflammation-Neuroinflammation, Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002, Basel, Switzerland.
authors.forename: Derya R
authors.initials: DR
authors.lastname: Shimshek
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Varani
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
authors.affiliation: Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131, Padova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Greggio
authors.affiliation: Department of Medical Sciences, Section of Pharmacology, and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy. m.morari@unife.it.
authors.forename: Michele
authors.identifier: 0000-0002-4601-4454
authors.initials: M
authors.lastname: Morari
mesh_terms: D000375:Aging / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D003342:Corpus Striatum / Q000378:metabolism* / Q000473:pathology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D055879:Gene Knock-In Techniques; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D020734:Parkinsonian Disorders / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D010766:Phosphorylation; D062706:Prodromal Symptoms; D013378:Substantia Nigra / Q000378:metabolism / Q000473:pathology; D050493:Vesicular Monoamine Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; C493453:Slc18a2 protein, mouse; C497605:Snca protein, mouse; D050493:Vesicular Monoamine Transport Proteins; D051844:alpha-Synuclein; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D004298:Dopamine
keywords: DAT; G2019S knock-in; LRRK2; Parkinson’s disease; VMAT2; α-synuclein