Abstract
Mutations in leucine‐rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro‐striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl‐evoked [3H]‐dopamine and glutamate release from superfused synaptosomes obtained from wild‐type and LRRK2 knock‐out, kinase‐dead or G2019S knock‐in mice was measured. Two structurally unrelated inhibitors, LRRK2‐IN‐1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2‐IN‐1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2‐IN‐1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock‐out and kinase‐dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock‐in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock‐in mice. We conclude that LRRK2‐IN‐1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine‐promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.
Highlights
Leucine-rich repeat kinase 2 (LRRK2) is a 2527 amino acid, multifunctional protein endowed with a kinase domain and a Ras of complex (ROC) domain with GTPase activity, surrounded by protein-protein interaction domains.[1,2] Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with familial late-onset 3,4 and sporadic 5 Parkinson's disease (PD)
We conclude that LRRK2-IN-1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, their effects vary depending on the nerve terminal examined
A synaptosomal preparation was used to investigate the role of LRRK2 in neurotransmitter release ([3H]-DA and GLU) in different brain areas, using two different LRRK2 inhibitors (IN-1 and GSK2578215A) and different mouse genotypes, that is, WT, KO, Kinase dead (KD), and G2019S KI mice (Table 2)
Summary
Leucine-rich repeat kinase 2 (LRRK2) is a 2527 amino acid, multifunctional protein endowed with a kinase domain and a Ras of complex (ROC) domain with GTPase activity, surrounded by protein-protein interaction domains.[1,2] Mutations in the LRRK2 gene are associated with familial late-onset 3,4 and sporadic 5 Parkinson's disease (PD). Synaptosomes represent a basic preparation of nerve endings, suitable for studying exocytosis since they preserve the release machinery (ATP-and Ca++- dependent release), express membrane and vesicular transporters, and expose autoreceptors In this preparation, the KCl-evoked neurotransmitter efflux relies on exocytotic Ca++-dependent and, partly, Na+-dependent mechanisms, whether spontaneous efflux is essentially non exocytotic.[40] the superfusion conditions adopted in this study ensure a rapid removal of the neurotransmitter from the medium, minimizing neurotransmitter uptake and autoreceptor activation,[41,42] which might confound the effect of the depolarizing stimulus and LRRK2 inhibitors on exocytosis. Since LRRK2 inhibitors are expected to be used in G2019S carriers first, their effects were investigated in synaptosomes from mice expressing the LRRK2 kinase-enhancing G2019S mutation (G2019S KI mice).[21,24,43] LRRK2 protein levels and kinase activity (pSer1292 and pSer[935] levels) were measured in striatal and cortical tissue lysates and synaptosomes, and target engagement of LRRK2 inhibitors assessed
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call