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Background and purpose Regulator of G-protein signal 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signaling. Here we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signaling and 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 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 upregulated RGS4 in the lesioned striatum. Conclusions and implications RGS4 physiologically inhibits NOP receptor signaling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 upregulation occurring during dyskinesia expression.

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familiar and sporadic 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 a-synuclein inclusions in striatal and nigral neurons were investigated in vivo. Methods: 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 a-synuclein inclusion in striatal MAP-positive and nigral TH-positive neurons. Results: No genotype differences in macroautophagy and chaperone-mediated autophagy 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/LC3-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, and GBA1 expression reduced in 12-month-old G2019S knock-in mice. Immunofluorescence revealed a dissociation between LC3B puncta accumulation and pSer129 a-synuclein inclusions in striatal neurons of kinase-dead and G2019S knock-in mice. Conclusions: We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy. In G2019S knock-in mice, pSer129 a-synuclein inclusions observed under basal conditions appear unrelated to autophagy impairment.

BackgroundMutations 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. Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering potential targets for neuroprotection are critically needed.A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causesan accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neuronsin vitro. MethodsBased on the hypothesisthat such KAR hyper-activation may contribute to the death of nigralDA 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. ResultsWe found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse model and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect was associated with rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. ConclusionsThis study provides novel evidence ofa 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 a neuroprotective therapy for ARJP.

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.

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 D1 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.

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin-NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin-NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, PCR analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus-pituitary-adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin-NOP system which is also altered in regions known to play a role in emotional aspects of pain.

Mutations in leucine‐rich repeat kinase 2 (LRRK2) gene have been pathogenically linked to Parkinson's disease, and pharmacological inhibition of LRRK2 is being pursued to tackle nigro‐striatal dopaminergic neurodegeneration. However, LRRK2 kinase inhibitors may have manifold actions, affecting not only pathological mechanisms in dopaminergic neurons but also physiological functions in nondopaminergic neurons. Therefore, we investigated whether LRRK2 kinase inhibitors differentially modulate dopamine and glutamate release from the mouse striatum and cerebral cortex. Spontaneous and KCl‐evoked [3H]‐dopamine and glutamate release from superfused synaptosomes obtained from wild‐type and LRRK2 knock‐out, kinase‐dead or G2019S knock‐in mice was measured. Two structurally unrelated inhibitors, LRRK2‐IN‐1 and GSK2578215A, were tested. LRRK2, phosphoSerine1292 and phosphoSerine935 LRRK2 levels were measured in all genotypes, and target engagement was evaluated by monitoring phosphoSerine935 LRRK2. LRRK2‐IN‐1 inhibited striatal glutamate but not dopamine release; GSK2578215A inhibited striatal dopamine and cortical glutamate but enhanced striatal glutamate release. LRRK2‐IN‐1 reduced striatal and cortical phosphoSerine935 levels whereas GSK2578215A inhibited only the former. Neither LRRK2 inhibitor affected neurotransmitter release in LRRK2 knock‐out and kinase‐dead mice; however, they facilitated dopamine without affecting striatal glutamate in G2019S knock‐in mice. GSK2578215A inhibited cortical glutamate release in G2019S knock‐in mice. We conclude that LRRK2‐IN‐1 and GSK2578215A modulate exocytosis by blocking LRRK2 kinase activity, although their effects vary depending on the nerve terminal examined. The G2019S mutation unravels a dopamine‐promoting action of LRRK2 inhibitors while blunting their effects on glutamate release, which highlights their positive potential for the treatment of PD, especially of LRRK2 mutation carriers.

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.

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.

The dynorphinergic system is involved in pain transmission at spinal level, where dynorphin exerts antinociceptive or pronociceptive effects, based on its opioid or non‐opioid actions. Surprisingly, little evidence is currently available concerning the supraspinal role of the dynorphinergic system in pain conditions. The present study aimed to investigate whether neuropathic pain is accompanied by prodynorphin (Pdyn) and κ‐opioid receptor (Oprk1) gene expression alterations in selected mouse brain areas. To this end, mice were subjected to chronic constriction injury of the right sciatic nerve and neuropathic pain behavioral signs were ascertained after 14 days. At this interval, a marked increase in Pdyn mRNA in the anterior cingulate cortex (ACC) and prefrontal cortex (PFC) was observed. Oprk1 gene expression was increased in the PFC, and decreased in the ACC and nucleus accumbens (NAc). No changes were observed in the other investigated regions. Because of the relationship between dynorphin and the brain‐derived neurotrophic factor, and the role of this neurotrophin in chronic pain‐related neuroplasticity, we investigated brain‐derived neurotrophic factor gene (Bdnf) expression in the areas showing Pdyn or Oprk1 mRNAs changes. Bdnf mRNA levels were increased in both the ACC and PFC, whereas no changes were assessed in the NAc. Present data indicate that the dynorphinergic system undergoes quite selective alterations involving the corticostriatal circuitry during neuropathic pain, suggesting a contribution to the negative affective component of pain. Moreover, parallel increases in Pdyn and Bdnf mRNA at cortical level suggest the occurrence of likely interactions between these systems in neuropathic pain maladaptive neuroplasticity., Dynorphinergic system undergoes selective alterations paralleled by Bdnf changes at corticostriatal level in chronic constriction injured mice.

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.

Tobacco smoking is particularly evident in individuals experiencing chronic pain. This complex relationship is poorly understood at both molecular and behavioral levels. Here, we describe experiments aimed at understanding whether a chronic pain state induces neuroadaptations into the brain or peripheral nerves that involve nicotinic acetylcholine receptors (nAChRs) and whether these neuroadaptations directly lead to increased vulnerability to nicotine addiction or to the development of coping strategies to relieve pain symptoms. We found that ligation of the rat L5 spinal nerve led to a dramatic downregulation in the mRNA expression levels of all nAChR subunits examined in dorsal root ganglia and a time-dependent downregulation of discrete subunits, particularly in the cingulate cortex and the amygdala. Spinal nerve ligation and sham-operated rats showed minor or no changes in patterns of acquisition and motivation for nicotine taking. Spinal nerve ligation rats also showed similar vulnerability to nicotine seeking as sham animals when reinstatement was induced by nicotine-associated cues, but failed to reinstate lever pressing when relapse was induced by nicotine priming. Spinal nerve ligation and sham rats were equally sensitive to nicotine-induced anxiety-like behavior and antinociception; however, nicotine produced a potent and long-lasting antiallodynic effect in spinal nerve ligation rats. These results demonstrate that chronic pain leads to plasticity of nAChRs that do not directly facilitate nicotine addictive behaviors. Instead, nicotine potently decreases allodynia, an effect that could lead to increased nicotine consumption in chronic pain subjects.

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.

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene ( Oprl1 ) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [ 35 S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. SIGNIFICANCE STATEMENT The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These knock-in mice have NOP receptors that function both in vitro and in vivo and have provided a detailed characterization of NOP receptors in brain, spinal cord, and DRG neurons. They appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function.

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

Background and Purpose The α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating nicotine reinforcement processes. AT-1001 has been recently described as a high-affinity and selective α3β4 nAChR antagonist that blocks nicotine self-administration in rats. The aim of this study was to investigate the mechanism of action underlying the nicotine-suppressive effects of AT-1001. Experimental Approach Effects of AT-1001 were determined using in vitro assays and rat models of nicotine addiction, and compared with varenicline. Key Results AT-1001 and its analogue AT-1012 were functionally selective as antagonists for α3β4 over α4β2 nAChRs, but not to the same extent as the binding selectivity, and had partial agonist activity at α3β4 nAChRs. In contrast, varenicline was a partial agonist at α4β2, a weak agonist at α3β4 and inhibited α4β2 at a much lower concentration than it inhibited α3β4 nAChRs. AT-1001 and varenicline also had very different in vivo properties. Firstly, AT-1001 did not exhibit reinforcing properties per se while varenicline was self-administered. Secondly, systemic treatment with AT-1001 did not induce reinstatement of nicotine seeking but rather attenuated reinstatement induced by varenicline, as well as nicotine. Finally, unlike varenicline, AT-1001 selectively blocked nicotine self-administration without altering alcohol lever pressing as assessed in an operant co-administration paradigm. Conclusions and Implications These findings describe a more complex AT-1001 in vitro profile than previously appreciated and provide further support for the potential of AT-1001 and congeners as clinically useful compounds for smoking cessation, with a mechanism of action distinct from currently available medications.

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.

Morphine is widely used for the treatment of severe acute and chronic pain, but long-term therapy rapidly leads to tolerance. Morphine effects are mediated by μ opioid receptor (MOP) activation as well as for fentanyl that, in contrast to morphine, induces less tolerance to anal- gesia. The mechanisms underlying opioid tolerance involve complex processes, such as MOP desensitization, internali- zation, and/or changes of gene expression. The develop- ment of morphine tolerance also involves adaptive changes of the anti-opioid nociceptin/orphanin FQ- nociceptin receptor system, as suggested by the reduc- tion of morphine tolerance in nociceptin opioid receptor (NOP) knockout mice. The aim of the present study was to investigate the MOP and NOP gene expression in the SH-SY5Y cells following morphine and fentanyl exposure. Results showed that cell exposure to 10 μM morphine for 5 h induced a significant decrease of MOP and NOP gene expression and that the MOP downregulation was reverted by the pretreatment with naloxone. Conversely, SH-SY5Y cells exposed to 0.1 and 1 μM fentanyl for 5 and 72 h showed a significant MOP upregulation, also reverted by naloxone pretreatment. Fentanyl induced no changes of NOP gene expression. The present findings showed a different effect by morphine and fentanyl on MOP mRNA levels that contributes to define the role of MOP gene expression changes in the mechanisms underlying the tolerance. Morphine also triggers an altered NOP-related signaling confirming that the nociceptin/orphanin FQ- nociceptin receptor system also plays a significant role in the development of morphine tolerance.