Anti-parkinsonian and anti-dyskinetic profiles of two novel potent and selective nociceptin/orphanin FQ receptor agonists

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

Anti-parkinsonian and anti-dyskinetic profiles of two novel potent and selective nociceptin/orphanin FQ receptor agonists

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

N/OFQ and the NOP receptor: a target with broad potential in Parkinson’s Disease

Il neuropeptide nocicettina/orfanina FQ (N / OFQ) ed il suo recettore (NOP) svolgono un ruolo patogenetico nella malattia di Parkinson (PD). Questo lavoro di tesi ha cercato di affrontare due questioni diverse e irrisolte legate al ruolo del sistema N/OFQ-NOP nel PD. Il primo, e forse più rilevante, è se N/OFQ endogena contribuisca alla morte dei neuroni dopaminergici della substantia nigra compacta, il segno distintivo del PD. Utilizzando modelli genetici ed etologici di PD, siamo stati in grado di dimostrare che la rimozione genetica ed il blocco farmacologico del recettore NOP hanno un impatto positivo sulla neurodegenerazione parkinsoniana, causando un risparmio significativo dei neuroni dopaminergici della sostanza nera e dei terminali dopaminergici striatali. Il secondo aspetto indagato è se la stimolazione del recettore NOP impedisce la sensibilizzazione dei neuroni striatali alla terapia con levodopa, un processo che è alla base dello sviluppo delle discinesie, una grave complicazione motoria associata alla terapia cronica con levodopa. Utilizzando il nuovo, potente e selettivo agonista pieno del recettore NOP AT-403, siamo stati in grado di dimostrare che la stimolazione del recettore NOP attenua lo sviluppo di movimenti anomaly involontari (il correlato delle discinesie nei roditori) indotti dalla somministrazione cronica di levodopa nei ratti emilesionati con 6-OHDA (un modello di PD), e attenua anche l'espressione dei movimenti discinetici indotti da somministrazione acuta di levodopa in ratti già discinetici. Tuttavia, questo effetto è stato accompagnato da una sedazione dose-dipendente, che restringe la finestra terapeutica del composto. Altri due potenti agonisti del recettore NOP testati nel corso dello studio, l’agonista completo AT-390 e l'agonista parziale AT-127, hanno confermato che l’effetto antidiscinetico e quello sedativo si sovrappongono parzialmente. In conclusione, questo studio conferma il ruolo di N/OFQ nel PD, e conseguentemente il recettore NOP come bersaglio promettente nella terapia del PD. Infatti, fornisce la prima evidenza di un effetto neuroprotettivo di un antagonista del recettore NOP, suggerendo come questa classe di composti potrebbe rivelarsi efficace non solo per alleviare i sintomi, ma anche come agenti in grado di modificare il corso della malattia.The neuropeptide Nociceptin/Orphanin FQ (N/OFQ) and its receptor (NOP) play a pathogenic role in Parkinson’s Disease (PD). This thesis work tried to address two different and unresolved questions related to the role of the N/OFQ-NOP system in PD. The first, and perhaps more relevant, is whether endogenous N/OFQ contributes to the death of dopamine neurons of substantia nigra compacta, the hallmark of PD. Using pathogenic and etiologic models of PD, we were able to demonstrate that genetic removal or pharmacological blockade of the NOP receptor had a beneficial impact on the parkinsonian neurodegeneration, causing a significant sparing of nigral dopamine neurons and striatal dopamine terminals. The second aspect investigated is whether NOP receptor stimulation prevents the sensitization of striatal neurons to levodopa, a process that underlies the development of dyskinesia, a severe motor complication associated with long term levodopa pharmacotherapy of PD. Using the novel, potent and selective NOP receptor full agonist AT-403, we were able to show that NOP receptor stimulation attenuates the development of abnormal involuntary movements (the rodent correlate of dyskinesia) induced by chronic levodopa administration in 6-OHDA hemilesioned rats (a model of PD), and also attenuates the expression of dyskinetic movements induced by acute levodopa challenge in already dyskinetic rats. Nonetheless, this effect was accompanied by a dose-dependent sedation which narrowed the therapeutic window of the compound. Other two potent NOP agonists tested along the study, the full agonists AT-390 and the partial agonist AT-127, confirmed that the antidyskinetic and the sedative dose-ranges partially overlap. In conclusion, this study confirms the role of N/OFQ in PD, and the NOP receptor as a promising target in PD therapy. It provides the first evidence of a neuroprotective/neurorescue effect of a NOP receptor antagonist, suggesting this class of compounds might prove effective not only as symptomatic but also disease-modifying agents in PD therapy

Parkinson's disease: no NOP, new hope

In conclusion, we feel that data obtained through a clinically-driven protocol in two different PD models (neurotoxic and etiologic) and animal species (mouse and rat), convey a solid proof-of-concept that NOP receptor antagonists protect/rescue DA neurons from PD-like neurodegeneration. Although more evidence needs to be collected to claim that endogenous N/OFQ contributes to DA cell loss also in human PD, NOP receptor antagonists hold promise as symptomatic and disease-modifying agents in PD therap

Differential regulation of striatal motor behavior and related cellular responses by dopamine D2L and D2S isoforms

The dopamine D2 receptor (D2R) is a major component of the dopamine system. D2R-mediated signaling in dopamine neurons is involved in the presynaptic regulation of dopamine levels. Postsynaptically, i.e., in striatal neurons, D2R signaling controls complex functions such as motor activity through regulation of cell firing and heterologous neurotransmitter release. The presence of two isoforms, D2L and D2S, which are generated by a mechanism of alternative splicing of the Drd2 gene, raises the question of whether both isoforms may equally control presynaptic and postsynaptic events. Here, we addressed this question by comparing behavioral and cellular responses of mice with the selective ablation of either D2L or D2S isoform. We establish that the presence of either D2L or D2S can support postsynaptic functions related to the control of motor activity in basal conditions. On the contrary, absence of D2S but not D2L prevents the inhibition of tyrosine hydroxylase phosphorylation and, thereby, of dopamine synthesis, supporting a major presynaptic role for D2S. Interestingly, boosting dopamine signaling in the striatum by acute cocaine administration reveals that absence of D2L, but not of D2S, strongly impairs the motor and cellular response to the drug, in a manner similar to the ablation of both isoforms. These results suggest that when the dopamine system is challenged, D2L signaling is required for the control of striatal circuits regulating motor activity. Thus, our findings show that D2L and D2S share similar functions in basal conditions but not in response to stimulation of the dopamine system

Age-dependent dopamine transporter dysfunction and Serine129 phospho-α-synuclein overload in G2019S LRRK2 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

Genetic and pharmacological evidence that endogenous nociceptin/orphanin FQ contributes to dopamine cell loss in Parkinson's disease

International audienceTo 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 4days 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

Age-dependent dopamine transporter dysfunction and Serine129 phospho-α-synuclein overload in G2019S LRRK2 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