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Mutations in the PRKN gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP). Harnessing this mutation to create an early-onset Parkinson's disease mouse model would provide a unique opportunity to clarify the mechanisms involved in the neurodegenerative process and lay the groundwork for the development of neuroprotective strategies. To this end, we created a knock-in mouse carrying the homozygous PrknR275W mutation, which is the missense mutation with the highest allelic frequency in PRKN patients. We evaluated the anatomical and functional integrity of the nigrostriatal dopamine (DA) pathway, as well as motor behaviour in PrknR275W mice of both sexes. We report here that PrknR275W mice show early DA neuron dysfunction, age-dependent loss of DA neurons in the substantia nigra, decreased DA content and stimulus-evoked DA release in the striatum, and progressive motor impairment. Together, these data show that the PrknR275W mouse recapitulates key features of ARJP. Thus, these studies fill a critical need in the field by introducing a promising new Parkinson's disease model in which to study causative mechanisms of the disease and test therapeutic strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are associated with familial and sporadic forms of Parkinson's disease (PD). Sporadic PD and LRRK2 PD share main clinical and neuropathological features, namely hypokinesia, degeneration of nigro-striatal dopamine neurons and α-synuclein aggregates in the form of Lewy bodies. Animals harboring the most common LRRK2 mutations, i.e. p.G2019S and p.R1441C/G, have been generated to replicate the parkinsonian phenotype and investigate the underlying pathogenic mechanisms. Disappointingly, however, LRRK2 rodents did not consistently phenocopy hypokinesia and nigro-striatal degeneration, or showed Lewy body-like aggregates. Instead, LRRK2 rodents manifested non-motor signs and dysregulated transmission at dopaminergic and non-dopaminergic synapses that are reminiscent of behavioral and functional network changes observed in the prodromal phase of the disease. LRRK2 rodents also manifested greater susceptibility to different parkinsonian toxins or stressors when subjected to dual-hit or multiple-hit protocols, confirming LRRK2 mutations as genetic risk factors. In conclusion, LRRK2 rodents represent a unique tool to identify the molecular mechanisms through which LRRK2 modulates the course and clinical presentations of PD and to study the interplay between genetic, intrinsic and environmental protective/risk factors in PD pathogenesis., Competing Interests: The authors declare that there are no competing interests associated with the manuscript., (© 2023 The Author(s).)

Background and Purpose: Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gα i/o and Gα q subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia., Experimental Approach: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured., Key Results: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum., Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression., Linked Articles: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Automatic analysis toolboxes are popular in brain image analysis, both in clinical and in preclinical practices. In this regard, we proposed a new toolbox for mouse PET-CT brain image analysis including a new Statistical Parametric Mapping-based template and a pipeline for image registration of PET-CT images based on CT images. The new templates is compatible with the common coordinate framework (CCFv3) of the Allen Reference Atlas (ARA) while the CT based registration step allows to facilitate the analysis of mouse PET-CT brain images. From the ARA template, we identified 27 volumes of interest that are relevant for in vivo imaging studies and provided binary atlas to describe them. We acquired 20 C57BL/6 mice with [ 18 F]FDG PET-CT, and 12 of them underwent 3D T2-weighted high-resolution MR scans. All images were elastically registered to the ARA atlas and then averaged. High-resolution MR images were used to validate a CT-based registration pipeline. The resulting method was applied to a mouse model of Parkinson's disease subjected to a test-retest study (n = 6) with the TSPO-specific radioligand [ 18 F]VC701. The identification of regions of microglia/macrophage activation was performed in comparison to the Ma and Mirrione template. The new toolbox identified 11 (6 after false discovery rate adjustment, FDR) brain sub-areas of significant [ 18 F]VC701 uptake increase versus the 4 (3 after FDR) macro-regions identified by the Ma and Mirrione template. Moreover, these 11 areas are functionally connected as found by applying the Mouse Connectivity tool of ARA. In conclusion, we developed a mouse brain atlas tool optimized for PET-CT imaging analysis that does not require MR. This tool conforms to the CCFv3 of ARA and could be applied to the analysis of mouse brain disease models., (© 2022. The Author(s).)

Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development., (© The Author(s) (2022). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The G2019S mutation in leucine rich-repeat kinase 2 (LRRK2) is a major cause of familial Parkinson's disease. We previously reported that G2019S knock-in mice manifest dopamine transporter dysfunction and phosphoSerine129 α-synuclein (pSer129 α-syn) immunoreactivity elevation at 12 months of age, which might represent pathological events leading to neuronal degeneration. Here, the time-dependence of these changes was monitored in the striatum of 6, 9, 12, 18 and 23-month-old G2019S KI mice and wild-type controls using DA uptake assay, Western analysis and immunohistochemistry. Western analysis showed elevation of membrane dopamine transporter (DAT) levels at 9 and 12 months of age, along with a reduction of vesicular monoamine transporter 2 (VMAT2) levels at 12 months. DAT uptake was abnormally elevated from 9 to up to 18 months. DAT and VMAT2 level changes were specific to the G2019S mutation since they were not observed in LRRK2 kinase-dead or knock-out mice. Nonetheless, dysfunctional DAT uptake was not normalized by acute pharmacological inhibition of LRRK2 kinase activity with MLi-2. Immunoblot analysis showed elevation of pSer129 α-syn levels in the striatum of 12-month-old G2019S KI mice, which, however, was not confirmed by immunohistochemical analysis. Instead, total α-syn immunoreactivity was found elevated in the striatum of 23-month-old LRRK2 knock-out mice. These data indicate mild changes in DA transporters and α-syn metabolism in the striatum of 12-month-old G2019S KI mice whose pathological relevance remains to be established.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with familial and sporadic forms of Parkinson's disease (PD), for which the LRRK2 locus itself represents a risk factor. Idiopathic and LRRK2-related PD share the main clinical and neuropathological features, thus animals harboring the most common LRRK2 mutations, i.e. G2019S and R1441C/G, have been generated to replicate the parkinsonian phenotype and investigate the underlying pathological mechanisms. Most LRRK2 rodent models, however, fail to show the main neuropathological hallmarks of the disease i.e. the degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of Lewy bodies or Lewy body-like aggregates of α-synuclein, lacking face validity. Rather, they manifest dysregulation in cellular pathways and functions that confer susceptibility to a variety of parkinsonian toxins/triggers and model the presymptomatic/premotor stages of the disease. Among such susceptibility factors, dysregulation of synaptic activity and proteostasis are evident in LRRK2 mutants. These abnormalities are also manifest in the PD brain and represent key events in the development and progression of the pathology. The present minireview covers recent articles (2018-2021) investigating the role of LRRK2 and LRRK2 mutants in the regulation of synaptic activity and autophagy-lysosomal pathway. These articles confirm a perturbation of synaptic vesicle endocytosis and glutamate release in LRRK2 mutants. Likewise, LRRK2 mutants show a marked impairment of selective forms of autophagy (i.e. mitophagy and chaperone-mediated autophagy) and lysosomal function, with minimal perturbations of nonselective autophagy. Thus, LRRK2 rodents might help understand the contribution of these pathways to PD., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The G2019S mutation of LRRK2 represents a risk factor for idiopathic Parkinson's disease. Here, we investigate whether LRRK2 kinase activity regulates susceptibility to the environmental toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). G2019S knock-in mice (bearing enhanced kinase activity) showed greater nigro-striatal degeneration compared to LRRK2 knock-out, LRRK2 kinase-dead and wild-type mice following subacute MPTP treatment. LRRK2 kinase inhibitors PF-06447475 and MLi-2, tested under preventive or therapeutic treatments, protected against nigral dopamine cell loss in G2019S knock-in mice. MLi-2 also rescued striatal dopaminergic terminal degeneration in both G2019S knock-in and wild-type mice. Immunoblot analysis of LRRK2 Serine935 phosphorylation levels confirmed target engagement of LRRK2 inhibitors. However, MLi-2 abolished phosphoSerine935 levels in the striatum and midbrain of both wild-type and G2019S knock-in mice whereas PF-06447475 partly reduced phosphoSerine935 levels in the midbrain of both genotypes. In vivo and ex vivo uptake of the 18-kDa translocator protein (TSPO) ligand [ 18 F]-VC701 revealed a similar TSPO binding in MPTP-treated wild-type and G2019S knock-in mice which was consistent with an increased GFAP striatal expression as revealed by Real Time PCR. We conclude that LRRK2 G2019S, likely through enhanced kinase activity, confers greater susceptibility to mitochondrial toxin-induced parkinsonism. LRRK2 kinase inhibitors are neuroprotective in this model., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 α-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous studies have pointed out that l-DOPA can interact with D1 or D2 receptors independent of its conversion to endogenous dopamine. The present study was set to investigate whether l-DOPA modulates dopamine release from striatal nerve terminals, using a preparation of synaptosomes preloaded with [ 3 H]DA. Levodopa (1 µM) doubled the K + -induced [ 3 H]DA release whereas the D2/D3 receptor agonist pramipexole (100 nM) inhibited it. The l-DOPA-evoked facilitation was mimicked by the D1 receptor agonist SKF38393 (30-300 nM) and prevented by the D1/D5 antagonist SCH23390 (100 nM) but not the DA transporter inhibitor GBR12783 (300 nM) or the aromatic l-amino acid decarboxylase inhibitor benserazide (1 µM). Higher l-DOPA concentrations (10 and 100 µM) elevated spontaneous [ 3 H]DA efflux. This effect was counteracted by GBR12783 but not SCH23390. Binding of [ 3 H]SCH23390 in synaptosomes (in test tubes) revealed a dense population of D1 receptors (2105 fmol/mg protein). Both SCH23390 and SKF38393 fully inhibited [ 3 H]SCH23390 binding (Ki 0.42 nM and 29 nM, respectively). l-DOPA displaced [ 3 H]SCH23390 binding maximally by 44% at 1 mM. This effect was halved by addition of GBR12935 and benserazide. We conclude that l-DOPA facilitates exocytotic [ 3 H]DA release through SCH23390-sensitive D1 receptors, independent of its conversion to DA. It also promotes non-exocytotic [ 3 H]DA release, possibly via conversion to DA and reversal of DA transporter. These data confirm that l-DOPA can directly interact with dopamine D1 receptors and might extend our knowledge of the neurobiological mechanisms underlying l-DOPA clinical effects., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. The LRRK2 G2019S mutation is one of the major determinants of familial Parkinson's disease cases and leads to late-onset Parkinson's disease with pleomorphic pathology, including α-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from patients with Parkinson's disease carrying the G2019S variant, and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Of note, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S bacterial artificial chromosome (BAC) mice. We suggest that LRRK2 G2019S pathological phosphorylation impacts on NSF biochemical properties, thus causing the formation of cytotoxic protein inclusions., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile parkinsonism (ARJP), a neurodegenerative disease characterized by early dysfunction and loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). No therapy is currently available to prevent or slow down the neurodegeneration in ARJP patients. Preclinical models are key to clarifying the early events that lead to neurodegeneration and reveal the potential of novel neuroprotective strategies. ParkinQ311X is a transgenic mouse model expressing in DA neurons a mutant parkin variant found in ARJP patients. This model was previously reported to show the neuropathological hallmark of the disease, i.e., the progressive loss of DA neurons. However, the early dysfunctions that precede neurodegeneration have never been investigated. Here, we analyzed SNc DA neurons in parkinQ311X mice and found early features of mitochondrial dysfunction, extensive cytoplasmic vacuolization, and dysregulation of spontaneous in vivo firing activity. These data suggest that the parkinQ311X mouse recapitulates key features of ARJP and provides a useful tool for studying the neurodegenerative mechanisms underlying the human disease and for screening potential neuroprotective drugs.

Basal expression of the P2X7 receptor (P2X7R) improves mitochondrial metabolism, Adenosine 5'-triphosphate (ATP) synthesis, and overall fitness of immune and non-immune cells. We investigated P2X7R contribution to energy metabolism and subcellular localization in fibroblasts (mouse embryo fibroblasts and HEK293 human fibroblasts), mouse microglia (primary brain microglia, and the N13 microglia cell line), and heart tissue. The P2X7R localizes to mitochondria, and its lack (1) decreases basal respiratory rate, ATP-coupled respiration, maximal uncoupled respiration, resting mitochondrial potential, mitochondrial matrix Ca 2+ level, (2) modifies expression pattern of oxidative phosphorylation enzymes, and (3) severely affects cardiac performance. Hearts from P2rx7 -deleted versus wild-type mice are larger, heart mitochondria smaller, and stroke volume, ejection fraction, fractional shortening, and cardiac output, are significantly decreased. Accordingly, the physical fitness of P2X7R-null mice is severely reduced. Thus, the P2X7R is a key modulator of mitochondrial energy metabolism and a determinant of physical fitness., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Physiological Society.)

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile Parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering targets for neuroprotection are critically needed. A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causes an accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neurons in vitro. Based on the hypothesis that such KAR hyperactivation may contribute to the death of nigral DA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. We found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse, and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect is associated with the rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. This study provides novel evidence of a causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of neuroprotective therapy for ARJP.

Acetylcholine muscarinic receptors (mAChRs) contribute to both the facilitation and inhibition of levodopa-induced dyskinesia operated by striatal cholinergic interneurons, although the receptor subtypes involved remain elusive. Cholinergic afferents from the midbrain also innervate the substantia nigra reticulata, although the role of nigral mAChRs in levodopa-induced dyskinesia is unknown. Here, we investigate whether striatal and nigral M1 and/or M4 mAChRs modulate dyskinesia and the underlying striato-nigral GABAergic pathway activation in 6-hydroxydopamine hemilesioned rats. Reverse microdialysis allowed to deliver the mAChR antagonists telenzepine (M1 subtype preferring), PD-102807 and tropicamide (M4 subtype preferring), as well as the selective M4 mAChR positive allosteric modulator VU0152100 in striatum or substantia nigra, while levodopa was administered systemically. Dyskinetic movements were monitored along with nigral GABA (and glutamate) and striatal glutamate dialysate levels, taken as neurochemical correlates of striato-nigral pathway and cortico-basal ganglia-thalamo-cortical loop activation. We observed that intrastriatal telenzepine, PD-102807 and tropicamide alleviated dyskinesia and inhibited nigral GABA and striatal glutamate release. This was partially replicated by intrastriatal VU0152100. The M2 subtype preferring antagonist AFDX-116, used to elevate striatal acetylcholine levels, blocked the behavioral and neurochemical effects of PD-102807. Intranigral VU0152100 prevented levodopa-induced dyskinesia and its neurochemical correlates whereas PD-102807 was ineffective. These results suggest that striatal, likely postsynaptic, M1 mAChRs facilitate dyskinesia and striato-nigral pathway activation in vivo. Conversely, striatal M4 mAChRs can both facilitate and inhibit dyskinesia, possibly depending on their localization. Potentiation of striatal and nigral M4 mAChR transmission leads to powerful multilevel inhibition of striato-nigral pathway and attenuation of dyskinesia., Competing Interests: Declaration of Competing Interest AB is postdoc at the University of Ferrara, CAP is PhD student at the University of Ferrara, MM is employed by the University of Ferrara. The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: l-DOPA-induced dyskinesia (LID) is considered a major complication in the treatment of Parkinson's disease (PD). Buspirone (5-HT 1A partial agonist) have shown promising results in the treatment of PD and LID, however no 5-HT-based treatment has been approved in PD. The present study was aimed to investigate how the substantia nigra pars reticulata (SNr) is affected by buspirone and whether it is a good target to study 5-HT antidyskinetic treatments., Experimental Approach: Buspirone was studied using in vivo single-unit, electrocorticogram, local field potential recordings along with microdialysis and immunohistochemistry in naïve/sham, 6-hydroxydopamine (6-OHDA)-lesioned or 6-OHDA-lesioned and l-DOPA-treated (6-OHDA/l-DOPA) rats., Key Results: Local buspirone inhibited SNr neuron activity in all groups. However, systemic buspirone reduced burst activity in 6-OHDA-lesioned rats (with or without l-DOPA treatment), whereas 8-OH-DPAT, a full 5-HT 1A agonist induced larger inhibitory effects in sham animals. Neither buspirone nor 8-OH-DPAT markedly modified the low-frequency oscillatory activity in the SNr or synchronization within the SNr with the cortex. In addition, local perfusion of buspirone increased GABA and glutamate release in the SNr of naïve and 6-OHDA-lesioned rats but no effect in 6-OHDA/l-DOPA rats. In the 6-OHDA/l-DOPA group, increased 5-HT transporter and decreased 5-HT 1A receptor expression was found., Conclusions and Implications: The effects of buspirone in SNr are influenced by dopamine loss and l-DOPA treatment. The present results suggest that the regulation of burst activity of the SNr induced by DA loss may be a good target to test new drugs for the treatment of PD and LID., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the appearance of α-synuclein insoluble aggregates known as Lewy bodies. Neurodegeneration is accompanied by neuroinflammation mediated by cytokines and chemokines produced by the activated microglia. Several studies demonstrated that such an inflammatory process is an early event, and contributes to oxidative stress and mitochondrial dysfunctions. α-synuclein fibrillization and aggregation activate microglia and contribute to disease onset and progression. Mutations in different genes exacerbate the inflammatory phenotype in the monogenic compared to sporadic forms of PD. Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) with selected radiopharmaceuticals allow in vivo imaging of molecular modifications in the brain of living subjects. Several publications showed a reduction of dopaminergic terminals and dopamine (DA) content in the basal ganglia, starting from the early stages of the disease. Moreover, non-dopaminergic neuronal pathways are also affected, as shown by in vivo studies with serotonergic and glutamatergic radiotracers. The role played by the immune system during illness progression could be investigated with PET ligands that target the microglia/macrophage Translocator protein (TSPO) receptor. These agents have been used in PD patients and rodent models, although often without attempting correlations with other molecular or functional parameters. For example, neurodegeneration and brain plasticity can be monitored using the metabolic marker 2-Deoxy-2-[ 18 F]fluoroglucose ([ 18 F]-FDG), while oxidative stress can be probed using the copper-labeled diacetyl-bis(N-methyl-thiosemicarbazone) ([Cu]-ATSM) radioligand, whose striatal-specific binding ratio in PD patients seems to correlate with a disease rating scale and motor scores. Also, structural and functional modifications during disease progression may be evaluated by Magnetic Resonance Imaging (MRI), using different parameters as iron content or cerebral volume. In this review article, we propose an overview of in vivo clinical and non-clinical imaging research on neuroinflammation as an emerging marker of early PD. We also discuss how multimodal-imaging approaches could provide more insights into the role of the inflammatory process and related events in PD development., (Copyright © 2020 Belloli, Morari, Murtaj, Valtorta, Moresco and Gilardi.)

Dopaminergic neuronal cell death, associated with intracellular α-synuclein (α-syn)-rich protein aggregates [termed "Lewy bodies" (LBs)], is a well-established characteristic of Parkinson's disease (PD). Much evidence, accumulated from multiple experimental models, has suggested that α-syn plays a role in PD pathogenesis, not only as a trigger of pathology but also as a mediator of disease progression through pathological spreading. Here, we have used a machine learning-based approach to identify unique signatures of neurodegeneration in monkeys induced by distinct α-syn pathogenic structures derived from patients with PD. Unexpectedly, our results show that, in nonhuman primates, a small amount of singular α-syn aggregates is as toxic as larger amyloid fibrils present in the LBs, thus reinforcing the need for preclinical research in this species. Furthermore, our results provide evidence supporting the true multifactorial nature of PD, as multiple causes can induce a similar outcome regarding dopaminergic neurodegeneration., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

To investigate whether the reversible MAO-B inhibitor and sodium channel blocker safinamide impairs glutamate release under parkinsonian conditions in vivo, and this effect is dependent on MAO-B inhibition, safinamide (and rasagiline as a comparator) were administered to 6-hydroxydopamine hemilesioned rats, a model of Parkinson's disease, and haloperidol-treated rats, a model of neuroleptic-induced parkinsonism. A microdialysis probe was implanted in the dopamine-depleted dorsolateral striatum, globus pallidus, subthalamic nucleus or substantia nigra reticulata of 6-hydroxydopamine hemilesioned rats. Glutamate and GABA release was stimulated by reverse dialysis of veratridine, and safinamide or rasagiline were acutely administered before veratridine at doses inhibiting MAO-B >50%. A microdialysis probe was implanted in the substantia nigra reticulata of naïve rats to monitor glutamate and GABA release following acute haloperidol and safinamide administration. Safinamide inhibited the veratridine-evoked glutamate release in the globus pallidus and subthalamic nucleus but not in the striatum and substantia nigra. Moreover, it reduced pallidal and nigral GABA release. Conversely, rasagiline failed to modify the veratridine-induced glutamate and GABA release in the basal ganglia. Safinamide also inhibited the haloperidol-induced nigral glutamate release. MAO-B inhibitors safinamide and rasagiline differ in their abilities to inhibit depolarization-evoked glutamate release in the basal ganglia of parkinsonian rats. The ineffectiveness of rasagiline suggests that MAO-B inhibition does not contribute to the antiglutamatergic activity of safinamide. The glutamate-inhibiting action of safinamide within the subthalamo-external pallidal loop, which shows abnormal activity in Parkinson's disease, might contribute to its therapeutic actions of improving motor performance without provoking troublesome dyskinesia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A novel series of C(3)-substituted piperdinylindoles were developed as nociceptin opioid receptor (NOP) partial agonists to explore a pharmacological hypothesis that NOP partial agonists would afford a dual pharmacological action of attenuating Parkinson's disease (PD) motor symptoms and development of levodopa-induced dyskinesias. SAR around the C-3 substituents investigated effects on NOP binding, intrinsic activity, and selectivity and showed that while the C(3)-substituted indoles are selective, high affinity NOP ligands, the steric, polar, and cationic nature of the C-3 substituents affected intrinsic activity to afford partial agonists with a range of efficacies. Compounds 4 , 5 , and 9 with agonist efficacies between 25% and 35% significantly attenuated motor deficits in the 6-OHDA-hemilesioned rat model of PD. Further, unlike NOP antagonists, which appear to worsen dyskinesia expression, these NOP partial agonists did not attenuate or worsen dyskinesia expression. The NOP partial agonists and their SAR reported here may be useful to develop nondopaminergic treatments for PD.

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP receptor) contribute to Parkinson's disease (PD) and motor complications associated with levodopa therapy. The N/OFQ-NOP receptor system is expressed in cortical and subcortical motor areas and, notably, in dopaminergic neurons of the substantia nigra compacta. Dopamine depletion, as in rodent models of PD results in up-regulation of N/OFQ transmission in the substantia nigra and down-regulation of N/OFQ transmission in the striatum. Consistent with this, NOP receptor antagonists relieve motor deficits in PD models by reinstating the physiological balance between excitatory and inhibitory inputs impinging on nigro-thalamic GABAergic neurons. NOP receptor antagonists also counteract the degeneration of nigrostriatal dopaminergic neurons, possibly by attenuating the excitotoxicity or modulating the immune response. Conversely, NOP receptor agonists attenuate levodopa-induced dyskinesia by attenuating the hyperactivation of striatal D 1 receptor signalling in neurons of the direct striatonigral pathway. The N/OFQ-NOP receptor system might represent a novel target in the therapy of PD., (© 2019 The British Pharmacological Society.)

Autophagy is a highly conserved process by which long-lived macromolecules, protein aggregates and dysfunctional/damaged organelles are delivered to lysosomes for degradation. Autophagy plays a crucial role in regulating protein quality control and cell homeostasis in response to energetic needs and environmental challenges. Indeed, activation of autophagy increases the life-span of living organisms, and impairment of autophagy is associated with several human disorders, among which neurodegenerative disorders of aging, such as Parkinson's disease. These disorders are characterized by the accumulation of aggregates of aberrant or misfolded proteins that are toxic for neurons. Since aging is associated with impaired autophagy, autophagy inducers have been viewed as a strategy to counteract the age-related physiological decline in brain functions and emergence of neurodegenerative disorders. Parkinson's disease is a hypokinetic, multisystemic disorder characterized by age-related, progressive degeneration of central and peripheral neuronal populations, associated with intraneuronal accumulation of proteinaceous aggregates mainly composed by the presynaptic protein α-synuclein. α-synuclein is a substrate of macroautophagy and chaperone-mediated autophagy (two major forms of autophagy), thus impairment of its clearance might favor the process of α-synuclein seeding and spreading that trigger and sustain the progression of this disorder. Genetic factors causing Parkinson's disease have been identified, among which mutations in the LRRK2 gene, which encodes for a multidomain protein encompassing central GTPase and kinase domains, surrounded by protein-protein interaction domains. Six LRRK2 mutations have been pathogenically linked to Parkinson's disease, the most frequent being the G2019S in the kinase domain. LRRK2-associated Parkinson's disease is clinically and neuropathologically similar to idiopathic Parkinson's disease, also showing age-dependency and incomplete penetrance. Several mechanisms have been proposed through which LRRK2 mutations can lead to Parkinson's disease. The present article will focus on the evidence that LRRK2 and its mutants are associated with autophagy dysregulation. Studies in cell lines and neurons in vitro and in LRRK2 knock-out, knock-in, kinase-dead and transgenic animals in vivo will be reviewed. The role of aging in LRRK2-induced synucleinopathy will be discussed. Possible mechanisms underlying the LRRK2-mediated control over autophagy will be analyzed, and the contribution of autophagy dysregulation to the neurotoxic actions of LRRK2 will be examined., (Copyright © 2019 Albanese, Novello and Morari.)

Parkinson's disease is characterized by the intracellular accumulation of α-synuclein which has been linked to early dopaminergic axonal degeneration. Identifying druggable targets that can promote axonal growth in cells overexpressing α-synuclein is important in order to develop strategies for early intervention. Class-IIa histone deacetylases (HDACs) have previously emerged as druggable targets, however, it is not known which specific class-IIa HDACs should be targeted to promote neurite growth in dopaminergic neurons. To provide insight into this, we used gene co-expression analysis to identify which, if any, of the class-IIa HDACs had a positive correlation with markers of dopaminergic neurons in the human substantia nigra. This revealed that two histone deacetylases, HDAC5 and HDAC9 , are co-expressed with TH, GIRK2 and ALDH1A1 in the human SN. We further found that HDAC5 and HDAC9 are expressed in dopaminergic neurons in the adult mouse substantia nigra. We show that siRNAs targeting HDAC5 or HDAC9 can promote neurite growth in SH-SY5Y cells, and that their pharmacological inhibition, using the drug MC1568, promoted neurite growth in cultured rat dopaminergic neurons. Moreover, MC1568 treatment upregulated the expression of the neurotrophic factor, BMP2 , and its downstream transcription factor, SMAD1 . In addition, MC1568 or siRNAs targeting HDAC5 or HDAC9 led to an increase in Smad-dependent GFP expression in a reporter assay. Furthermore, MC1568 treatment of cultured rat dopaminergic neurons increased cellular levels of phosphorylated Smad1, which was prevented by the BMP receptor inhibitor, dorsomorphin. Dorsomorphin treatment prevented the neurite growth-promoting effects of siRNAs targeting HDAC5, as did overexpression of dominant-negative Smad4 or of the inhibitory Smad7, demonstrating a functional link to BMP signaling. Supplementation with BMP2 prevented the neurite growth-inhibitory effects of nuclear-restricted HDAC5. Finally, we report that siRNAs targeting HDAC5 or HDAC9 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein and that MC1568 protected cultured rat dopaminergic neurons against the neurotoxin, MPP + . These findings establish HDAC5 and HDAC9 as novel regulators of BMP-Smad signaling, that additionally may be therapeutic targets worthy of further exploration in iPSC-derived human DA neurons and in vivo models of Parkinson's disease., (Copyright © 2019 Mazzocchi, Wyatt, Mercatelli, Morari, Morales-Prieto, Collins, Sullivan and O’Keeffe.)

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 [ 3 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., Competing Interests: DRS is employed by Novartis Pharma. All other Authors declare no competing interests.

Nociceptin/Orphanin FQ (N/OFQ) and its NOP receptor are highly expressed in motor areas of the rodent, nonhuman, and human primate brain, such as primary motor cortex, thalamus, globus pallidus, striatum, and substantia nigra. Endogenous N/OFQ negatively regulates motor behavior and dopamine transmission through NOP receptors expressed by dopaminergic neurons of the substantia nigra compacta. Consistent with the existence of an N/OFQ tone over dopaminergic transmission, blockade of NOP receptor antagonists increases striatal dopamine release. In this chapter, we will review the evidence linking the N/OFQ-NOP receptor system to Parkinson's disease (PD). We will first discuss data showing that the central N/OFQ-NOP receptor system undergoes plastic changes in different basal ganglia nuclei following dopamine depletion. Then we will show that NOP receptor antagonists relieve motor deficits in different rodent and nonhuman primate models of PD. Mechanistically, NOP receptor blockade in substantia nigra reticulata results in rebalancing of the inhibitory GABAergic and excitatory glutamatergic inputs impinging on nigro-thalamic GABAergic neurons, leading to thalamic disinhibition. We will also present data showing that, in addition to motor symptoms, N/OFQ also plays a role in the parkinsonian neurodegeneration. In fact, NOP receptor antagonists possess neuroprotective/neurorescue properties in in vitro and in vivo models of PD.

Fibrillization of α-synuclein is instrumental for the development of Parkinson's disease (PD), thus modulating this process can have profound impact on disease initiation/progression. Here, the impact of the p.G2019S mutation of leucine-rich repeat kinase 2 (LRRK2), which is most frequently associated with familial and sporadic PD, on α-synuclein pathology was investigated. G2019S knock-in mice and wild-type controls were injected with a recombinant adeno-associated viral vector serotype 2/9 (AAV2/9) overexpressing human mutant p.A53T α-synuclein (AAV2/9-hα-syn). Control animals were injected with AAV2/9 carrying green fluorescent protein. Motor behavior, transgene expression, α-syn and pSer129 α-syn load, number of nigral dopamine neurons and density of striatal dopaminergic terminals were evaluated. To investigate the effect of aging, experiments were performed in 3- and 12-month-old mice, evaluated 20 and 12 weeks after virus injection, respectively. hα-syn overexpression induced progressive motor deficits, loss of nigral dopaminergic neurons and striatal terminals, and appearance of proteinase K-resistant aggregates of pSer129 α-syn in both young and old mice. Although no genotype difference was observed in 3-month-old mice, degeneration of nigral dopaminergic neurons was higher in 12-month-old G2019S knock-in mice compared with age-matched wild-type controls (-55% vs -39%, respectively). Consistently, a two-fold higher load of pSer129 α-syn aggregates was found in 12-month-old G2019S knock-in mice. We conclude that G2019S LRRK2 facilitates α-synucleinopathy and degeneration of nigral dopaminergic neurons, and that aging is a major determinant of this effect., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Safinamide (Xadago) is a novel dual-mechanism drug that has been approved in the European Union and United States as add-on treatment to levodopa in Parkinson's disease therapy. In addition to its selective and reversible monoamine oxidase B inhibition, safinamide through use-dependent sodium channel blockade reduces overactive glutamatergic transmission in basal ganglia, which is believed to contribute to motor symptoms and complications including levodopa-induced dyskinesia (LID). The present study investigated the effects of safinamide on the development of LID in 6-hydroxydopamine (6-OHDA)-lesioned rats, evaluating behavioral, molecular, and neurochemical parameters associated with LID appearance. 6-OHDA-lesioned rats were treated with saline, levodopa (6 mg/kg), or levodopa plus safinamide (15 mg/kg) for 21 days. Abnormal involuntary movements, motor performance, molecular composition of the striatal glutamatergic synapse, glutamate, and GABA release were analyzed. In the striatum, safinamide prevented the rearrangement of the subunit composition of N -methyl-d-aspartate receptors and the levodopa-induced increase of glutamate release associated with dyskinesia without affecting the levodopa-stimulated motor performance and dyskinesia. Overall, these findings suggest that the striatal glutamate-modulating component of safinamide's activity may contribute to its clinical effects, where its long-term use as levodopa add-on therapy significantly improves motor function and "on" time without troublesome dyskinesia., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia., Methods: To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling., Results: We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming., Conclusions: Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.

Among genetic abnormalities identified in Parkinson's disease (PD), mutations of the leucine-rich repeat kinase2 (LRRK2) gene, such as the G2019S missense mutation linked to enhanced kinase activity, are the most common. While the complex role of LRRK2 has not been fully elucidated, evidence that mutated kinase activity affects synaptic transmission has been reported. Thus, our aim was to explore possible early alterations of neurotransmission produced by the G2019S LRRK2 mutation in PD. We performed electrophysiological patch-clamp recordings of striatal spiny projection neurons (SPNs) in the G2019S-Lrrk2 knock-in (KI) mouse model of PD, in D1994S kinase-dead (KD), Lrrk2 knock-out (KO) and wild-type (WT) mice. In G2019S Lrrk2 KI mice, basal spontaneous glutamatergic transmission, synaptic facilitation, and NMDA/AMPA ratios were unchanged, whereas the stimulation of dopamine (DA) D2 receptor by quinpirole reduced the spontaneous and evoked excitatory postsynaptic currents (EPSC). Quinpirole reduced the EPSC amplitude of SPNs in KI but not in KD, KO and WT mice, suggesting that the enhanced LRRK2 kinase activity induced by the G2019S mutation is associated with the observed functional alteration of SPNs synaptic transmission. The effect of quinpirole was mediated by a phospholipase C (PLC)-dependent release of endocannabinoid, with subsequent activation of presynaptic cannabinoid receptor 1 and reduced release of glutamate. The key role of DA D2 receptor in reducing glutamatergic output in our LRRK2 genetic model of PD further supports the use of DA agonists in the treatment of early PD patients with LRRK2 mutations to counteract the disease progression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The solute concentration in crystallization processes is generally estimated by observing properties of the liquid phase. Here, a novel method for online estimation of the change in the solute concentration caused by seeded batch crystallization or dissolution of a population of crystals in suspension is presented. The method is based on multiprojection imaging to track variations in the total solid volume of the population, thus enabling inference of the solute concentration through the mass conservation constraint. The solute concentration estimates obtained in this way are validated by using them to measure the solubilities of β l-glutamic acid and vanillin in water within certain temperature ranges and comparing them to literature data. The presented method shows promise in estimating the solute concentration reliably under circumstances where employing conventional techniques is challenging.

Background and Purpose: We previously showed that nociceptin/orphanin FQ opioid peptide (NOP) receptor agonists attenuate the expression of levodopa-induced dyskinesia in animal models of Parkinson's disease. We now investigate the efficacy of two novel, potent and chemically distinct NOP receptor agonists, AT-390 and AT-403, to improve Parkinsonian disabilities and attenuate dyskinesia development and expression., Experimental Approach: Binding affinity and functional efficacy of AT-390 and AT-403 at the opioid receptors were determined in radioligand displacement assays and in GTPγS binding assays respectively, conducted in CHO cells. Their anti-Parkinsonian activity was evaluated in 6-hydroxydopamine hemi-lesioned rats whereas the anti-dyskinetic properties were assessed in 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa. The ability of AT-403 to inhibit the D 1 receptor-induced phosphorylation of striatal ERK was investigated., Key Results: AT-390 and AT-403 selectively improved akinesia at low doses and disrupted global motor activity at higher doses. AT-403 palliated dyskinesia expression without causing sedation in a narrow therapeutic window, whereas AT-390 delayed the appearance of abnormal involuntary movements and increased their duration at doses causing sedation. AT-403 did not prevent the priming to levodopa, although it significantly inhibited dyskinesia on the first day of administration. AT-403 reduced the ERK phosphorylation induced by SKF38393 in vitro and by levodopa in vivo., Conclusions and Implications: NOP receptor stimulation can provide significant albeit mild anti-dyskinetic effect at doses not causing sedation. The therapeutic window, however, varies across compounds. AT-403 could be a potent and selective tool to investigate the role of NOP receptors in vivo., (© 2017 The British Pharmacological Society.)

Parkinson's disease (PD) is a neurodegenerative disorder in which genetic and environmental factors synergistically lead to loss of midbrain dopamine (DA) neurons. Mutation of leucine-rich repeated kinase2 (Lrrk2) genes is responsible for the majority of inherited familial cases of PD and can also be found in sporadic cases. The pathophysiological role of this kinase has to be fully understood yet. Hyperactivation of Lrrk2 kinase domain might represent a predisposing factor for both enhanced striatal glutamatergic release and mitochondrial vulnerability to environmental factors that are observed in PD. To investigate possible alterations of striatal susceptibility to mitochondrial dysfunction, we performed electrophysiological recordings from the nucleus striatum of a G2019S Lrrk2 mouse model of PD, as well as molecular and morphological analyses of G2019S Lrrk2-expressing SH-SY5Y neuroblastoma cells. In G2019S mice, we found reduced striatal DA levels, according to the hypothesis of alteration of dopaminergic transmission, and increased loss of field potential induced by the mitochondrial complex I inhibitor rotenone. This detrimental effect is reversed by the D2 DA receptor agonist quinpirole via the inhibition of the cAMP/PKA intracellular pathway. Analysis of mitochondrial functions in G2019S Lrrk2-expressing SH-SY5Y cells revealed strong rotenone-induced oxidative stress characterized by reduced Ca 2+ buffering capability and ATP synthesis, production of reactive oxygen species, and increased mitochondrial fragmentation. Importantly, quinpirole was able to prevent all these changes. We suggest that the G2019S-Lrrk2 mutation is a predisposing factor for enhanced striatal susceptibility to mitochondrial dysfunction induced by exposure to mitochondrial environmental toxins and that the D2 receptor stimulation is neuroprotective on mitochondrial function, via the inhibition of cAMP/PKA intracellular pathway. We suggest new possible neuroprotective strategies for patients carrying this genetic alteration based on drugs specifically targeting Lrrk2 kinase domain and mitochondrial functionality.

Safinamide has been recently approved as an add-on to levodopa therapy for Parkinson disease. In addition to inhibiting monoamine oxidase type B, it blocks sodium channels and modulates glutamate (Glu) release in vitro. Since this property might contribute to the therapeutic action of the drug, we undertook the present study to investigate whether safinamide inhibits Glu release also in vivo and whether this effect is consistent across different brain areas and is selective for glutamatergic neurons. To this aim, in vivo microdialysis was used to monitor the spontaneous and veratridine-induced Glu and GABA release in the hippocampus and basal ganglia of naive, awake rats. Brain levels of safinamide were measured as well. To shed light on the mechanisms underlying the effect of safinamide, sodium currents were measured by patch-clamp recording in rat cortical neurons. Safinamide maximally inhibited the veratridine-induced Glu and GABA release in hippocampus at 15 mg/kg, which reached free brain concentrations of 1.89-1.37 µ M. This dose attenuated veratridine-stimulated Glu (but not GABA) release in subthalamic nucleus, globus pallidus, and substantia nigra reticulata, but not in striatum. Safinamide was ineffective on spontaneous neurotransmitter release. In vitro, safinamide inhibited sodium channels, showing a greater affinity at depolarized (IC 50 = 8 µ M) than at resting (IC 50 = 262 µ M) potentials. We conclude that safinamide inhibits in vivo Glu release from stimulated nerve terminals, likely via blockade of sodium channels at subpopulations of neurons with specific firing patterns. These data are consistent with the anticonvulsant and antiparkinsonian actions of safinamide and provide support for the nondopaminergic mechanism of its action., (Copyright © 2018 The Author(s).)

Visually guided finger movements include online feedback of current effector position to guide target approach. This visual feedback may be scaled or otherwise distorted by unpredictable perturbations. Although adjustments to visual feedback scaling have been studied before, the underlying brain activation differences between upscaling (visual feedback larger than real movement) and downscaling (feedback smaller than real movement) are currently unknown. Brain activation differences between upscaling and downscaling might be expected because within-trial adjustments during upscaling require corrective backwards accelerations, whereas correcting for downscaling requires forward accelerations. In this behavioural and fMRI study we investigated adjustments during up- and downscaling in a target-directed finger flexion-extension task with real-time visual feedback. We found that subjects made longer and more complete within-trial corrections for downscaling perturbations than for upscaling perturbations. The finger task activated primary motor (M1) and somatosensory (S1) areas, premotor and parietal regions, basal ganglia, and cerebellum. General scaling effects were seen in the right pre-supplementary motor area, dorsal anterior cingulate cortex, inferior parietal lobule, and dorsolateral prefrontal cortex. Stronger activations for down- than for upscaling were observed in M1, supplementary motor area (SMA), S1 and anterior cingulate cortex. We argue that these activation differences may reflect differing online correction for upscaling vs. downscaling during finger flexion-extension., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

Antipsychotic drugs are currently used in clinical practice for a variety of mental disorders. Among them, clozapine is the most effective medication for treatment-resistant schizophrenia and is most helpful in controlling aggression and the suicidal behavior in schizophrenia and schizoaffective disorder. Although clozapine is associated with a low likelihood of extrapyramidal symptoms and other neurological side effects, it is well known for the weight gain and metabolic side effects, which expose the patient to a greater risk of cardiovascular disorders and premature death, as well as psychosocial issues, leading to non-adherence to therapy. The mechanisms underlying these iatrogenic metabolic disorders are still controversial. We have therefore investigated the in vivo effects of the selective PKCβ inhibitor, ruboxistaurin (LY-333531), in a preclinical model of long-term clozapine-induced weight gain. Cell biology, biochemistry, and behavioral tests have been performed in wild-type and PKCβ knockout mice to investigate the contribution of endogenous PKCβ and its pharmacological inhibition to the psychomotor effects of clozapine. Finally, we also shed light on a novel aspect of the mechanism underlying the clozapine-induced weight gain, demonstrating that the clozapine-dependent PKCβ activation promotes the inhibition of the lipid droplet-selective autophagy process. This paves the way to new therapeutic approaches to this serious complication of clozapine therapy.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

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.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like neuropeptide that binds and signals through a G-protein-coupled receptor called the N/OFQ peptide (NOP) receptor. N/OFQ and the NOP receptor are expressed in the midbrain and have been implicated in the pathogenesis of Parkinson's disease (PD). Genetic removal of the N/OFQ precursor partially protects midbrain dopaminergic neurons from 1-methyl-4-phenylpyridine-induced toxicity, suggesting that endogenous N/OFQ may be detrimental to dopaminergic neurons. However, whether N/OFQ directly affects the survival and growth of dopaminergic neurons is unknown. Here, we show that N/OFQ has a detrimental effect on the survival of dopaminergic neurons and the growth of their axons in primary cultures of the E14 rat ventral mesencephalon. N/OFQ potentiates the effects of the neurotoxins 6-hydroxydopamine and 1-methyl-4-phenylpyridinium through p38-MAPK signalling. We also show that like α-synuclein, there is a significant reduction in N/OFQ messenger RNA (mRNA) expression in the midbrain of patients with Parkinson's disease. These results demonstrate for the first time that N/OFQ is detrimental to the survival and growth of dopaminergic neurons and that its expression is altered in the midbrain of patients with Parkinson's disease.

Computer interaction via visually guided hand or finger movements is a ubiquitous part of daily computer usage in work or gaming. Surprisingly, however, little is known about the performance effects of using virtual limb representations versus simpler cursors. In this study 26 healthy right-handed adults performed cued index finger flexion-extension movements towards an on-screen target while wearing a data glove. They received each of four different types of real-time visual feedback: a simple circular cursor, a point light pattern indicating finger joint positions, a cartoon hand and a fully shaded virtual hand. We found that participants initiated the movements faster when receiving feedback in the form of a hand than when receiving circular cursor or point light feedback. This overall difference was robust for three out of four hand versus circle pairwise comparisons. The faster movement initiation for hand feedback was accompanied by a larger movement amplitude and a larger movement error. We suggest that the observed effect may be related to priming of hand information during action perception and execution affecting motor planning and execution. The results may have applications in the use of body representations in virtual reality applications.

To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinson's disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP(-/-)) mice, and the selective and potent small molecule NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP(-/-) mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP(-/-) mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinson's disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinson's disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones., (Copyright © 2016. Published by Elsevier Inc.)

Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein that modulates signal transduction at dopamine receptors, and also activates mammalian target of rapamycin complex 1 (mTORC1). Rhes binding to mTORC1 is hypothesized to play a role in motor disorders such as levodopa-induced dyskinesia. Here, we investigate the behavioral and in vivo neurocircuitry changes associated with genetic deletion of Rhes or inhibition of mTORC1 signaling in the mouse model of levodopa-induced dyskinesia. 6-Hydroxydopamine-hemilesioned Rhes knockout mice and wild-type littermates were chronically treated with levodopa. In parallel, 6-hydroxydopamine-hemilesioned naïve mice were chronically treated with levodopa or levodopa plus rapamycin, to block mTORC1 pathway activation. Dyskinetic movements were monitored during levodopa treatment along with motor activity on the rotarod. Finally, dyskinetic mice underwent microdialysis probe implantation in the dopamine-depleted striatum and ipsilateral substantia nigra reticulata, and GABA and glutamate levels were monitored upon acute challenge with levodopa. Both Rhes knockouts and rapamycin-treated mice developed less dyskinesia than controls, although only rapamycin-treated mice fully preserved rotarod performance on levodopa. Levodopa elevated nigral GABA and glutamate in controls but not in Rhes knockouts or rapamycin-treated mice. Levodopa also stimulated striatal glutamate in controls and Rhes knockouts but not in rapamycin-treated mice. We conclude that both genetic deletion of Rhes and pharmacological blockade of mTORC1 significantly attenuate dyskinesia development by reducing the sensitization of striato-nigral medium-sized spiny neurons to levodopa. However, mTORC1 blockade seems to provide a more favorable behavioral outcome and a wider effect on neurochemical correlates of dyskinesia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Preclinical and clinical evidence that the serotonergic system plays a major role in levodopa-induced dyskinesias has been provided. Selective serotonin (5-hydroxytryptamine; 5-HT) 5-HT1A or 5-HT1B receptor agonists, and, very recently, the mixed 5-HT1A /5-HT1B receptor agonist, eltoprazine, proved effective in inhibiting L-dopa-induced dyskinesias in experimental animals and parkinsonian patients. Here, we investigate the mechanisms underlying this effect., Methods: Microdialysis was employed in 6-hydroxydopamine-hemilesioned rats chronically treated with L-dopa alone or in combination with eltoprazine. Gamma-aminobutyric acid (GABA) and glutamate levels were monitored on L-dopa in the dopamine-depleted striatum and ipsilateral SNr. Motor activity on the rotarod was assessed, both off and on L-dopa. Western blot was used to quantify ex vivo striatal levels of phosphorylated extracellular signal-regulated kinase 1 and 2. Striatal and nigral amino acid levels, as well as striatal dopamine levels, were also monitored in L-dopa-primed dyskinetic rats acutely challenged with L-dopa and eltoprazine., Results: Eltoprazine attenuated the development and expression of dyskinesias, preserving motor coordination on the rotarod. Eltoprazine prevented the rise of nigral amino acids and striatal glutamate levels, as well as the increase in striatal phosphorylated extracellular signal-regulated kinase 1 and 2, associated with dyskinesias. However, eltoprazine did not affect the L-dopa-induced increase in striatal dopamine., Conclusions: Eltoprazine inhibits the sensitization of striatonigral medium-sized GABA spiny neurons (the direct pathway) to L-dopa and their overactivation associated with dyskinesias appearance. Activation of 5-HT1A and 5-HT1B receptors regulating striatal glutamate transmission, but not striatal ectopic dopamine release, might underlie the symptomatic effect of eltoprazine., (© 2015 International Parkinson and Movement Disorder Society.)

Introduction: Germline silencing of the PD-related protein LRRK2 does not alter glutamate or dopamine release in adult mice, but some exploratory abnormalities have been reported with ageing. Contrastingly, high levels of human LRRK2 cause locomotor alterations and cognitive deficits accompanied by reduced striatal dopamine levels, with the latter also observed in G2019S mutant mice. Comparative cognitive and motor behavioral testing of LRRK2 KO, overexpressor and mutant overexpressor mice has not previously been reported., Methods: Parallel, comparative behavioral characterization was performed assessing motor and cognitive abilities. Striatal antisense oligonucleotide injections were conducted to investigate the effects of acute LRRK2 silencing on behavior and dopamine fiber density. Striatal synaptosomes prepared from hG2019S mice assessed vesicular release of dopamine and its sensitivity to D2 autoreceptor stimulation., Results: Genetic ablation of LRRK2 has no long-term consequences on motor or cognitive function. Consistently, no effects on behavior or dopaminergic fiber density were observed following acute striatal silencing. Conversely, 12-month OE mice show persistent locomotor deficits and worsening of cognitive abilities; whereas, hG2019S mice display early hyperactivity and effective learning and memory that progress to decreased motor and cognitive deficits at older ages. The G2019S mutation does not affect vesicular dopamine release, but decreases its sensitivity to D2-mediated inhibition., Conclusion: LRRK2 silencing is well tolerated in mouse, arguing PD does not result from LRRK2 loss of function. High levels of WT and G2019S LRRK2 produce similar but temporally distinct phenotypes, potentially modeling different stages of disease progression. The data implicate gain of LRRK2 function in the pathogenesis of PD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa (L-dopa) therapy for Parkinson's disease (PD). L-dopa-induced dyskinesia (LID) are ultimately experienced by the vast majority of patients. In addition, psychiatric conditions often manifested as compulsive behaviours, are emerging as a serious problem in the management of L-dopa therapy. The present review attempts to provide an overview of our current understanding of dyskinesia and other L-dopa-induced dysfunctions, a field that dramatically evolved in the past twenty years. In view of the extensive literature on LID, there appeared a critical need to re-frame the concepts, to highlight the most suitable models, to review the central nervous system (CNS) circuitry that may be involved, and to propose a pathophysiological framework was timely and necessary. An updated review to clarify our understanding of LID and other L-dopa-related side effects was therefore timely and necessary. This review should help in the development of novel therapeutic strategies aimed at preventing the generation of dyskinetic symptoms., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many current therapies target G protein coupled receptors (GPCR), transporters, or ion channels. In addition to directly targeting these proteins, disrupting the protein-protein interactions that localize or regulate their function could enhance selectivity and provide unique pharmacologic actions. Regulators of G protein signaling (RGS) proteins, especially RGS4, play significant roles in epilepsy and Parkinson's disease. Thiadiazolidinone (TDZD) inhibitors of RGS4 are nanomolar potency blockers of the biochemical actions of RGS4 in vitro. Here, we demonstrate the substantial selectivity (8- to >5000-fold) of CCG-203769 for RGS4 over other RGS proteins. It is also 300-fold selective for RGS4 over GSK-3β, another target of this class of chemical scaffolds. It does not inhibit the cysteine protease papain at 100 μM. CCG-203769 enhances Gαq-dependent cellular Ca(2+) signaling in an RGS4-dependent manner. TDZD inhibitors also enhance Gαi-dependent δ-OR inhibition of cAMP production in SH-SY-5Y cells, which express endogenous receptors and RGS4. Importantly, CCG-203769 potentiates the known RGS4 mechanism of Gαi-dependent muscarinic bradycardia in vivo. Furthermore, it reverses raclopride-induced akinesia and bradykinesia in mice, a model of some aspects of the movement disorder in Parkinson's disease. A broad assessment of compound effects revealed minimal off-target effects at concentrations necessary for cellular RGS4 inhibition. These results expand our understanding of the mechanism and specificity of TDZD RGS inhibitors and support the potential for therapeutic targeting of RGS proteins in Parkinson's disease and other neural disorders.

By using multi-probe microdialysis we previously demonstrated that endogenous glutamate differentially regulates the activity of the striatal output pathways in vivo, through N-methyl-d-aspartate (NMDA) receptors containing the GluN2A or GluN2B subunits. Using the same approach, we presently investigate whether reverse dialysis of NMDA in the striatum differentially affects GABA release in the striatum and in striatal target areas, i.e. globus pallidus (GP) and substantia nigra reticulata (SNr). Moreover, we ask whether this control is altered under parkinsonian conditions. Intrastriatal NMDA perfusion (10 min) evoked GABA release more potently in SNr (1-100 μM) than in other regions (10-100 μM), suggesting preferential control over striato-nigral projection neurons. Intrastriatal NMDA more potently stimulated glutamate levels in the striatum (1-100 μM) and SNr (1-10 μM) than in GP (10 μM). Striatal dopamine denervation with 6-hydroxydopamine caused a leftward shift in the NMDA concentration-response curve. Intrastriatal NMDA elevated GABA levels at 0.1 μM (all regions) and 1 μM (striatum and GP only), but not at higher concentrations, indicating that, compared to naïve animals, the GABA response in SNr was attenuated. Attenuation of the glutamate response was also observed in SNr (NMDA effective only at 0.1 μM). Conversely, the glutamate response in GP was widened (NMDA effective in the 0.1-1 μM range). We conclude that NMDA preferentially stimulates the activity of the striato-nigral direct pathway under physiological conditions. In Parkinson's disease, dopamine loss compromises the NMDA ability to stimulate striato-nigral neurons, thus shifting the NMDA control towards the striato-pallidal ones.

Objective: Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates., Methods: We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson's disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed., Results: Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID., Interpretation: Our results suggest that Ras-GRF1 is a promising target for LID therapy based on Ras-ERK signaling inhibition in the striatum.