Age-dependent dopamine transporter dysfunction and Serine129 phospho-?-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 ?-synuclein or ?-synuclein bound to DOPAL (a toxic metabolite of dopamine). However, Serine129-phosphorylated ?-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 ?-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.
Project description:Mutations in leucine-rich repeat kinase 2 (<i>LRRK2</i>) 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 [<sup>3</sup>H]-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.
Project description:PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.
Project description:LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson's disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose toward disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of miniature excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using two-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.
Project description:The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson's disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (?-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether ?-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify ?-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal ?-synucleinopathy.
Project description:Sleep disturbances co-occur with and precede the onset of motor symptoms in Parkinson's disease (PD). We evaluated sleep fragmentation and thalamocortical sleep spindles in mice expressing the p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) gene, one of the most common genetic forms of PD. Thalamocortical sleep spindles are oscillatory events that occur during slow-wave sleep that are involved in memory consolidation. We acquired data from electrocorticography, sleep behavioral measures, and a rotarod-based motor enrichment task in 28 LRRK2-G2019S knock-in mice and 27 wild-type controls (8-10 month-old males). Sleep was more fragmented in LRRK2-G2019S mice; sleep bouts were shorter and more numerous, even though total sleep time was similar to controls. LRRK2-G2019S animals expressed more sleep spindles, and individual spindles were longer in duration than in controls. We then chronically administered the LRRK2-inhibitor MLi-2 in-diet to n = 12 LRRK2-G2019S and n = 15 wild-type mice for a within-subject analysis of the effects of kinase inhibition on sleep behavior and physiology. Treatment with MLi-2 did not impact these measures. The data indicate that the LRRK2-G2019S mutation could lead to reduced sleep quality and altered sleep spindle physiology. This suggests that sleep spindles in LRRK2-G2019S animals could serve as biomarkers for underlying alterations in sleep networks resulting from the LRRK2-G2019S mutation, and further evaluation in human LRRK2-G2019S carriers is therefore warranted.
Project description:Mutations in glucocerebrosidase 1 (GBA1) represent the most prevalent risk factor for Parkinson's disease. The molecular mechanisms underlying the link between GBA1 mutations and Parkinson's disease are incompletely understood. We analysed two aged (24-month-old) Gba1 mouse models, one carrying a knock-out mutation and the other a L444P knock-in mutation. A significant reduction of glucocerebrosidase activity was associated with increased total alpha-synuclein accumulation in both these models. Gba1 mutations alone did not alter the number of nigral dopaminergic neurons nor striatal dopamine levels. We then investigated the effect of overexpression of human alpha-synuclein in the substantia nigra of aged (18 to 21-month-old) L444P Gba1 mice. Following intraparenchymal injections of human alpha-synuclein carrying viral vectors, pathological accumulation of phosphorylated alpha-synuclein occurred within the transduced neurons. Stereological counts of nigral dopaminergic neurons revealed a significantly greater cell loss in Gba1-mutant than wild-type mice. These results indicate that Gba1 deficiency enhances neuronal vulnerability to neurodegenerative processes triggered by increased alpha-synuclein expression.
Project description:<h4>Background</h4>Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated α-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD, as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how α-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses.<h4>Methods</h4>Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages, dendritic cells, or microglia, and exposed to well-characterized human or mouse α-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies, and myeloid cell responses to α-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with α-synuclein fibrils and microglia in Boyden chambers.<h4>Results</h4>α-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells, α-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast, LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to α-synuclein fibrils. Corroborating these results, LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery, distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to α-synuclein fibrils. In primary cultured macrophages, LRRK2 kinase inhibition dampens α-synuclein fibril and microglia-stimulated chemotaxis.<h4>Conclusions</h4>Pathologic α-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain.
Project description:Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.
Project description:BACKGROUND:Multiple missense mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with familial forms of late onset Parkinson's disease (PD), the most common age-related movement disorder. The dysfunction of dopamine transmission contributes to PD-related motor symptoms. Interestingly, LRRK2 is more abundant in the dopaminoceptive striatal spiny projection neurons (SPNs) compared to the dopamine-producing nigrostriatal dopaminergic neurons. Aging is the most important risk factor for PD and other neurodegenerative diseases. However, whether LRRK2 modulates the aging of SPNs remains to be determined. METHODS:We conducted RNA-sequencing (RNA-seq) analyses of striatal tissues isolated from Lrrk2 knockout (Lrrk2-/-) and control (Lrrk2+/+) mice at 2 and 12?months of age. We examined SPN nuclear DNA damage and epigenetic modifications; SPN nuclear, cell body and dendritic morphology; and the locomotion and motor skill learning of Lrrk2+/+ and Lrrk2-/- mice from 2 to 24?months of age. Considering the strength of cell cultures for future mechanistic studies, we also performed preliminary studies in primary cultured SPNs derived from the Lrrk2+/+ and Lrrk2-/- mice as well as the PD-related Lrrk2 G2019S and R1441C mutant mice. RESULTS:Lrrk2-deficiency accelerated nuclear hypertrophy and induced dendritic atrophy, soma hypertrophy and nuclear invagination in SPNs during aging. Additionally, increased nuclear DNA damage and abnormal histone methylations were also observed in aged Lrrk2-/- striatal neurons, together with alterations of molecular pathways involved in regulating neuronal excitability, genome stability and protein homeostasis. Furthermore, both the PD-related Lrrk2 G2019S mutant and LRRK2 kinase inhibitors caused nuclear hypertrophy, while the Lrrk2 R1441C mutant and ?-Aminobutyric acid type A receptor (GABA-AR) inhibitors promoted nuclear invagination in the cultured SPNs. On the other hand, inhibition of neuron excitability prevented the formation of nuclear invagination in the cultured Lrrk2-/- and R1441C SPNs. CONCLUSIONS:Our findings support an important physiological function of LRRK2 in maintaining nuclear structure integrity and genomic stability during the normal aging process, suggesting that PD-related LRRK2 mutations may cause the deterioration of neuronal structures through accelerating the aging process.
Project description:Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.